Two euAGAMOUS Genes Control C-Function in Medicago truncatula

C-function MADS-box transcription factors belong to the AGAMOUS (AG) lineage and specify both stamen and carpel identity and floral meristem determinacy. In core eudicots, the AG lineage is further divided into two branches, the euAG and PLE lineages. Functional analyses across flowering plants strongly support the idea that duplicated AG lineage genes have different degrees of subfunctionalization of the C-function. The legume Medicago truncatula contains three C-lineage genes in its genome: two euAG genes (MtAGa and MtAGb) and one PLENA-like gene (MtSHP). This species is therefore a good experimental system to study the effects of gene duplication within the AG subfamily. We have studied the respective functions of each euAG genes in M. truncatula employing expression analyses and reverse genetic approaches. Our results show that the M. truncatula euAG- and PLENA-like genes are an example of subfunctionalization as a result of a change in expression pattern. MtAGa and MtAGb are the only genes showing a full C-function activity, concomitant with their ancestral expression profile, early in the floral meristem, and in the third and fourth floral whorls during floral development. In contrast, MtSHP expression appears late during floral development suggesting it does not contribute significantly to the C-function. Furthermore, the redundant MtAGa and MtAGb paralogs have been retained which provides the overall dosage required to specify the C-function in M. truncatula.

Related papers:

title: Flower development in Arabidopsis: there is more to it than learning your ABCs.
abstract: The field of Arabidopsis flower development began in the early 1980s with the initial description of several mutants including apetala1, apetala2, and agamous that altered floral organ identity (Koornneef and van der Veen, Theor Appl Genet 58:257-263, 1980; Koornneef et al., J Hered 74:265-272, 1983). By the end of the 1980s, these mutants were receiving more focused attention to determine precisely how they affected flower development (Komaki et al., Development 104:195-203, 1988; Bowman et al., Plant Cell 1:37-52, 1989). In the last quarter century, impressive progress has been made in characterizing the gene products and molecular mechanisms that control the key events in flower development. In this review, we briefly summarize the highlights of work from the past 25 years but focus on advances in the field in the last several years.

title: Floral organ identity: 20 years of ABCs.
abstract: One of the early successes of the application of molecular genetics to study plant development was the discovery of a series of genes that act together, in an apparently simple combinatorial model, to specify the identity of the different organs of a flower. Widely known as the ABC model, this framework for understanding has been investigated and modified over the course of the last two decades. The cast list of genes has been defined and, as other chapters in this volume will show, great progress has been made in understanding how they are regulated, how they act together, what they do and how they have contributed to the evolution of the flower in its varied forms. In this introductory review to the volume we will review the derivation and elaboration of the most current version of the ABC model, highlighting the modifications that have been necessary to ensure it fits the available experimental data. We will highlight the remaining difficulties in fitting the current model to the experimental data and propose a further modification to enable it to regain its applicability.

title: The flowering of Arabidopsis flower development.
abstract: Flowers come in a variety of colors, shapes and sizes. Despite this variety, flowers have a very stereotypical architecture, consisting of a series of sterile organs surrounding the reproductive structures. Arabidopsis, as the premier model system for molecular and genetic analyses of plant development, has provided a wealth of insights into how this architecture is specified. With the advent of the completion of the Arabidopsis genome sequence a decade ago, in combination with a rich variety of forward and reverse genetic strategies, many of the genes and regulatory pathways controlling flower initiation, patterning, growth and differentiation have been characterized. A central theme that has emerged from these studies is the complexity and abundance of both positive and negative feedback loops that operate to regulate different aspects of flower formation. Presumably, this considerable degree of feedback regulation serves to promote a robust and stable transition to flowering, even in the face of genetic or environmental perturbations. This review will summarize recent advances in defining the genes, the regulatory pathways, and their interactions, that underpin how the Arabidopsis flower is formed.

title: MADS-box genes and floral development: the dark side.
abstract: The origin of the flower during evolution has been a crucial step in further facilitating plants to colonize a wide range of different niches on our planet. The >250 000 species of flowering plants existing today display an astonishing diversity in floral architecture. For this reason, the flower is a very attractive subject for evolutionary developmental (evo-devo) genetics studies. Research during the last two decades has provided compelling evidence that the origin and functional diversification of MIKC(c) MADS-box transcription factors has played a critical role during evolution of flowering plants. As master regulators of floral organ identity, MADS-box proteins are at the heart of the classic ABC model for floral development. Despite the enormous progress made in the field of floral development, there still remain aspects that are less well understood. Here we highlight some of the dark corners within our current knowledge on MADS-box genes and flower development, which would be worthwhile investigating in more detail in future research. These include the general question of to what extent MADS-box gene functions are conserved between species, the function of TM8-clade MADS-box genes which so far have remained uncharacterized, the divergence within the A-function, and post-transcriptional regulation of the ABC-genes.

title: Complementary floral homeotic phenotypes result from opposite orientations of a transposon at the plena locus of antirrhinum
abstract: Recessive mutations at the plena (ple) locus result in a homeotic conversion of sex organs to sterile perianth organs in flowers of Antirrhinum majus. A complementary phenotype, in which sex organs replace sterile organs, is conferred by semidominant ovulata mutations. The ple locus was identified and isolated using a homologous gene, agamous from Arabidopsis, as a probe. The expression of ple is normally restricted to the inner two whorls of the flower, where sex organs develop. However, in ovulata mutants, ple is expressed ectopically in the outer two whorls of the flower and in vegetative organs. These mutants correspond to gain-of-function alleles of ple, suggesting that ple is sufficient for promoting sex organ development within the context of the flower. The plena and ovulata phenotypes result from opposite orientations of the transposon Tam3 inserted in the large intron of ple.

title: Assessing the redundancy of MADS-box genes during carpel and ovule development
abstract: Carpels are essential for sexual plant reproduction because they house the ovules and subsequently develop into fruits that protect, nourish and ultimately disperse the seeds. The AGAMOUS (AG) gene is necessary for plant sexual reproduction because stamens and carpels are absent from ag mutant flowers. However, the fact that sepals are converted into carpelloid organs in certain mutant backgrounds even in the absence of AG activity indicates that an AG-independent carpel-development pathway exists. AG is a member of a monophyletic clade of MADS-box genes that includes SHATTERPROOF1 (SHP1), SHP2 and SEEDSTICK (STK), indicating that these four genes might share partly redundant activities. Here we show that the SHP genes are responsible for AG-independent carpel development. We also show that the STK gene is required for normal development of the funiculus, an umbilical-cord-like structure that connects the developing seed to the fruit, and for dispersal of the seeds when the fruit matures. We further show that all four members of the AG clade are required for specifying the identity of ovules, the landmark invention during the course of vascular plant evolution that enabled seed plants to become the most successful group of land plants.

title: SHATTERPROOF MADS-box genes control seed dispersal in Arabidopsis
abstract: The fruit, which mediates the maturation and dispersal of seeds, is a complex structure unique to flowering plants. Seed dispersal in plants such as Arabidopsis occurs by a process called fruit dehiscence, or pod shatter. Few studies have focused on identifying genes that regulate this process, in spite of the agronomic value of controlling seed dispersal in crop plants such as canola. Here we show that the closely related SHATTERPROOF (SHP1) and SHATTERPROOF2 (SHP2 ) MADS-box genes are required for fruit dehiscence in Arabidopsis. Moreover, SHP1 and SHP2 are functionally redundant, as neither single mutant displays a novel phenotype. Our studies of shp1 shp2 fruit, and of plants constitutively expressing SHP1 and SHP2, show that these two genes control dehiscence zone differentiation and promote the lignification of adjacent cells. Our results indicate that further analysis of the molecular events underlying fruit dehiscence may allow genetic manipulation of pod shatter in crop plants.

title: PLENA and FARINELLI: redundancy and regulatory interactions between two Antirrhinum MADS‐box factors controlling flower development
abstract: We report the discovery of an Antirrhinum MADS‐box gene, FARINELLI (FAR), and the isolation of far mutants by a reverse genetic screen. Despite striking similarities between FAR and the class C MADS‐box gene PLENA (PLE), the phenotypes of their respective mutants are dramatically different. Unlike ple mutants, which show homeotic conversion of reproductive organs to perianth organs and a loss of floral determinacy, far mutants have normal flowers which are partially male‐sterile. Expression studies of PLE and FAR, in wild‐type and mutant backgrounds, show complex interactions between the two genes. Double mutant analysis reveals an unexpected, redundant negative control over the B‐function MADS‐box genes. This feature of the two Antirrhinum C‐function‐like genes is markedly different from the control of the inner boundary of the B‐function expression domain in Arabidopsis, and we propose and discuss a model to account for these differences. The difference in phenotypes of mutants in two highly related genes illustrates the importance of the position within the regulatory network in determining gene function.

title: Patterns of gene duplication and functional evolution during the diversification of the AGAMOUS subfamily of MADS box genes in angiosperms.
abstract: Members of the AGAMOUS (AG) subfamily of MIKC-type MADS-box genes appear to control the development of reproductive organs in both gymnosperms and angiosperms. To understand the evolution of this subfamily in the flowering plants, we have identified 26 new AG-like genes from 15 diverse angiosperm species. Phylogenetic analyses of these genes within a large data set of AG-like sequences show that ancient gene duplications were critical in shaping the evolution of the subfamily. Before the radiation of extant angiosperms, one event produced the ovule-specific D lineage and the well-characterized C lineage, whose members typically promote stamen and carpel identity as well as floral meristem determinacy. Subsequent duplications in the C lineage resulted in independent instances of paralog subfunctionalization and maintained functional redundancy. Most notably, the functional homologs AG from Arabidopsis and PLENA (PLE) from Antirrhinum are shown to be representatives of separate paralogous lineages rather than simple genetic orthologs. The multiple subfunctionalization events that have occurred in this subfamily highlight the potential for gene duplication to lead to dissociation among genetic modules, thereby allowing an increase in morphological diversity.

title: The major clades of MADS-box genes and their role in the development and evolution of flowering plants.
abstract: MADS-box genes encode a family of transcription factors which control diverse developmental processes in flowering plants ranging from root to flower and fruit development. Sequencing of (almost) the complete Arabidopsis genome enabled the identification of (almost) all of the Arabidopsis MADS-box genes. MADS-box genes have been divided in two large groups, termed type I and type II genes. The type II genes comprise the MEF2-like genes of animals and fungi and the MIKC-type genes of plants. The majority of MIKC-type genes are of the MIKC(c)-type, which includes all plant MADS-box genes for which expression patterns or mutant phenotypes are known. By phylogeny reconstruction, almost all of the MIKC(c)-type genes can be subdivided into 12 major gene clades, each clade comprising 1-6 paralogs from Arabidopsis and putative orthologs from other seed plants. Here we first briefly describe the deep branching of the MADS-box gene tree to place the MIKC(c)-type genes into an evolutionary context. For every clade of MIKC(c)-type genes we then review what is known about its members from Arabidopsis and well-studied members from other phylogenetically informative plant species. By gene sampling and phylogeny reconstructions we provide minimal estimates for the ages of the different clades. It turns out that 7 of the 12 major gene clades, i.e., AG-, AGL6-, AGL12-, DEF+GLO- (B), GGM13- (B(s)), STMADS11- and TM3-like genes very likely existed already in the most recent common ancestor of angiosperms and gymnosperms about 300MYA. Three of the other clades, i.e., AGL2-, AGL17-, and SQUA-like genes, existed at least already in the most recent common ancestor of monocots and eudicots about 200 MYA. Only for two gene clades, AGL15-like genes (2 genes in Arabidopsis) and FLC-like genes (6 genes) members from plants other than Brassicaceae have not been reported yet. Similarly, only one ancient clade known from other flowering plant species, TM8-like genes, is not represented in Arabidopsis. These findings reveal that the diversity of MADS-box genes in Arabidopsis is rather ancient and representative for other flowering plants. Our studies may thus help to predict the set of MADS-box genes in all other flowering plants, except for relatively young paralogs. For the different gene clades we try to identify ancestral and derived gene functions and review the importance of these clades for seed plant development and evolution. We put special emphasis on gene clades for which insights into their importance has rapidly increased just recently.

title: The evolution of floral homeotic gene function.
abstract: Plant MADS-box genes encode transcriptional regulators that are critical for a number of developmental processes. In the angiosperms (the flowering plants), these include the specification of floral organ identities, flowering time and fruit development. It appears that the MADS box gene family has undergone considerable gene duplication and sequence divergence within the angiosperms. Here I discuss the possibility that these events have allowed the recruitment of these genes to new developmental pathways in particular angiosperm lineages. Recent analyses of sequence changes, expression patterns and, in a few cases, gene function are beginning to provide tantalizing evidence for deciphering when and how such genetic diversification has led to particular morphological innovations. In the future, comparative studies of large numbers of species will be required to assess the extent of such variation as well as to fully understand the mechanisms by which evolution of these developmental regulators has played a role in shaping new morphologies.

title: Conservation and divergence in the AGAMOUS subfamily of MADS‐box genes: evidence of independent sub‐ and neofunctionalization events
abstract: The MADS‐box gene AGAMOUS (AG) plays a key role in determining floral meristem and organ identities. We identified three AG homologs, EScaAG1, EScaAG2, and EScaAGL11 from the basal eudicot Eschscholzia californica (California poppy). Phylogenetic analyses indicate that EScaAG1 and EScaAG2 are recent paralogs within the AG clade, independent of the duplication in ancestral core eudicots that gave rise to the euAG and PLENA (PLE) orthologs. EScaAGL11 is basal to core eudicot AGL11 orthologs in a clade representing an older duplication event after the divergence of the angiosperm and gymnosperm lineages. Detailed in situ hybridization experiments show that expression of EScaAG1 and EScaAG2 is similar to AG; however, both genes appear to be expressed earlier in floral development than described in the core eudicots. A thorough examination of available expression and functional data in a phylogenetic context for members of the AG and AGL11 clades reveals that gene expression has been quite variable throughout the evolutionary history of the AG subfamily and that ovule‐specific expression might have evolved more than twice. Although sub‐ and neofunctionalization are inferred to have occurred following gene duplication, functional divergence among orthologs is evident, as is convergence, among paralogs sampled from different species. We propose that retention of multiple AG homologs in several paralogous lineages can be explained by the conservation of ancestral protein activity combined with evolutionarily labile regulation of expression in the AG and AGL11 clades such that the collective functions of the AG subfamily in stamen and carpel development are maintained following gene duplication.

title: AGL1-AGL6, an Arabidopsis gene family with similarity to floral homeotic and transcription factor genes.
abstract: The predicted products of floral homeotic genes, AGAMOUS (AG) from Arabidopsis thaliana and DEFICIENS A (DEF A) from Antirrhinum majus, have been shown previously to share strong sequence similarity with transcription factors from humans (SRF) and yeast (MCM1). The conserved sequence between these proteins is localized within a domain known to be necessary for the DNA binding and for the dimerization of SRF. We have isolated six new genes from A. thaliana, AGL1-AGL6, which also have this conserved sequence motif. On the basis of the sequence comparison between the AG and AGL genes, they can be assigned to two subfamilies of a large gene family. RNA dot blot analysis indicates that five of these genes (AGL1, AGL2, AGL4, AGL5, and AGL6) are preferentially expressed in flowers. In addition, in situ RNA hybridization experiments with AGL1 and AGL2 show that their mRNAs are detected in some floral organs but not in others. Our results suggest that these genes may act to control many steps of Arabidopsis floral morphogenesis. In contrast, the AGL3 gene is expressed in vegetative tissues as well as in flowers, suggesting that it functions in a broader range of tissues. We discuss possible roles of this gene family during the evolution of flowers.

title: Temporal relationship between the transcription of two Arabidopsis MADS box genes and the floral organ identity genes.
abstract: MADS box genes play important roles in specifying floral meristem and floral organ identity. We characterized the temporal and spatial expression patterns of two members of this gene family, AGL4 and AGL5 (for AGAMOUS [AG]-like). AGL4 RNA initially accumulates after the onset of expression of the floral meristem identity genes but before the onset of expression of the floral organ identity genes. AGL4 is, therefore, a putative target of the floral meristem identity genes and/or a potential regulator of the floral organ identity genes. AGL5 is initially expressed early in carpel development, shortly after the onset of AG expression. The loss of AGL5 expression in flowers of ag mutants, the activation of AGL5 by ectopic expression of AG, and the specific binding of AG to an element in the AGL5 promoter identify AGL5 as a putative direct target of AG. Our study provides possible links between the establishment of floral meristem and floral organ identity as well as subsequent steps in flower development.

title: A new role for the SHATTERPROOF genes during Arabidopsis gynoecium development.
abstract: Gynoecium development is a complex process which is regulated by key factors that control the spatial formation of the apical, medial and basal parts. SHATTERPROOF1 (SHP1) and SHP2, two closely related MADS-box genes, redundantly control the differentiation of the dehiscence zone and promote the lignification of adjacent cells. Furthermore, SHP1 and SHP2 have shown to play an important role in ovule identity determination. The present work identifies a new function for these two genes in promoting stigma, style and medial tissue development. This new role was discovered by combining the shp1 shp2 double mutant with the aintegumenta (ant) and crabs claw (crc) mutants. In quadruple mutant flowers, the inner whorl is composed of unfused carpels which lack almost completely apical and medial tissues, a phenotype similar to the previously reported fil ant and lug ant double mutants.

title: Functional analyses of AGAMOUS family members in Nicotiana benthamiana clarify the evolution of early and late roles of C-function genes in eudicots.
abstract: The C-function, according to the ABC model of floral organ identity, is required for stamen and carpel development and to provide floral meristem determinacy. Members of the AG lineage of the large MADS box gene family specify the C-function in a broadly conserved manner in angiosperms. In core eudicots, two sub-lineages co-exist, euAG and PLE, which have been extensively characterized in Antirrhinum majus and Arabidopsis thaliana, where strong sub-functionalization has led to highly divergent contributions of the respective paralogs to the C-function. Various scenarios have been proposed to reconstruct the evolutionary history of the euAG and PLE lineages in eudicots, but detailed functional analyses of the roles of these genes in additional representative species to validate evolutionary hypotheses are scarce. Here, we report functional characterization of euAG- and PLE-like genes in Nicotiana benthamiana through expression analyses and phenotypic characterization of the defects caused by their specific down-regulation. We show that both paralogs redundantly contribute to the C-function in this species, providing insights on the likely evolution of these gene lineages following divergence of the major groups within the eudicots (rosids and asterids). Moreover, we have demonstrated a conserved role for the PLE-like genes in controlling fruit dehiscence, which strongly supports the ancestral role of PLE-like genes in late fruit development and suggests a common evolutionary origin of late developmental processes in dry (dehiscent) and fleshy (ripening) fruits.

title: Role of petunia pMADS3 in determination of floral organ and meristem identity, as revealed by its loss of function.
abstract: pMADS3, a petunia class C gene, is the candidate homologue of Arabidopsis AGAMOUS (AG), which is involved in the specification of stamens and carpels. We report the characterization of loss-of-function phenotype of pMADS3 that resulted from silencing of this gene. Silencing of pMADS3 resulted in homeotic conversion of stamens into petaloid structures, whereas the carpels were only weakly affected. Ectopic secondary inflorescences emerged from the interstamenal region in the third whorl, which is unique and has not been reported for any class C gene of other plant species. Third-order inflorescences emerged at corresponding positions in the third whorl of inner flowers of secondary inflorescences, indicating reiterative conversion of parts of the floral meristem into inflorescence meristem. On the basis of phenotypic analysis of the pMADS3-silenced plants, we propose that pMADS3 is involved in determination of floral organ and floral meristem identity in petunia. Two hybrid studies in yeast showed that PMADS3 protein interacted specifically with FBP2, a candidate homologue of Arabidopsis SEPALLATA3 (SEP3). The evidence presented here suggest that a complex involving PMADS3 and FBP2 is responsible for specification of organ identity in the third whorl.

title: Functional diversification of AGAMOUS lineage genes in regulating tomato flower and fruit development
abstract: AGAMOUS clade genes encode MADS box transcription factors that have been shown to play critical roles in many aspects of flower and fruit development in angiosperms. Tomato possesses two representatives of this lineage, TOMATO AGAMOUS (TAG1) and TOMATO AGAMOUS-LIKE1 (TAGL1), allowing for an analysis of diversification of function after gene duplication. Using RNAi (RNA interference) silencing, transgenic tomato lines that specifically down-regulate either TAGL1 or TAG1 transcript accumulation have been produced. TAGL1 RNAi lines show no defects in stamen or carpel identity, but show defects in fruit ripening. In contrast TAG1 RNAi lines show defects in stamen and carpel development. In addition TAG1 RNAi lines produce red ripe fruit, although they are defective in determinacy and produce ectopic internal fruit structures. e2814, an EMS- (ethyl methane sulphonate) induced mutation that is temperature sensitive and produces fruit phenotypes similar to that of TAG1 RNAi lines, was also characterized. Neither TAG1 nor TAGL1 expression is disrupted in the e2814 mutant, suggesting that the gene corresponding to the e2814 mutant represents a distinct locus that is likely to be functionally downstream of TAG1 and TAGL1. Based on these analyses, possible modes by which these gene duplicates have diversified in terms of their functions and regulatory roles are discussed.

title: Isolation of the tomato AGAMOUS gene TAG1 and analysis of its homeotic role in transgenic plants.
abstract: To understand the details of the homeotic systems that govern flower development in tomato and to establish the ground rules for the judicious manipulation of this floral system, we have isolated the tomato AGAMOUS gene, designated TAG1, and examined its developmental role in antisense and sense transgenic plants. The AGAMOUS gene of Arabidopsis is necessary for the proper development of stamens and carpels and the prevention of indeterminate growth of the floral meristem. Early in flower development, TAG1 RNA accumulates uniformly in the cells fated to differentiate into stamens and carpels and later becomes restricted to specific cell types within these organs. Transgenic plants that express TAG1 antisense RNA display homeotic conversion of third whorl stamens into petaloid organs and the replacement of fourth whorl carpels with pseudocarpels bearing indeterminate floral meristems with nested perianth flowers. A complementary phenotype was observed in transgenic plants expressing the TAG1 sense RNA in that first whorl sepals were converted into mature pericarpic leaves and sterile stamens replaced the second whorl petals.

title: Structure and expression of duplicate AGAMOUS orthologues in poplar
abstract: To investigate the homeotic systems underlying floral development in a dioecious tree, and to provide tools for the manipulation of floral development, we have isolated two Populus trichocarpa genes, PTAG1 and PTAG2, homologous to the Arabidopsis floral homeotic gene AGAMOUS (AG). PTAG1 and PTAG2 are located on separate linkage groups, but their non-coding regions are highly similar, consistent with a phylogenetically recent duplication. Intron/exon structure is conserved in relation to AG and the Antirrhinum AG orthologue, PLENA (PLE), and low-stringency Southern analysis demonstrated the absence of additional genes in the poplar genome with significant PTAG1/2 homology. PTAG1 and PTAG2 exhibit an AG-like floral expression pattern, and phylogenetic analysis of the AG subfamily strongly supports evolutionary orthology to C-class organ identity genes. The high degree of similarity shared by PTAG1 and PTAG2 in both sequence (89% amino acid identity) and expression indicates that they are unlikely to be functionally associated with specification of tree gender. Unexpectedly, PTAG transcripts were consistently detected in vegetative tissues.

title: Expression of multiple AGAMOUS-like genes in male and female flowers of cucumber (Cucumis sativus L.).
abstract: Members of the MADS-box gene family control reproductive development in higher plants. In cucumber, floral development exhibits several interesting features related to a genetically determined sex-expression mechanism, that affects the differentiation of male and female flowers. In this study, three cDNA homologues of the homeotic gene AGAMOUS have been cloned from early-stage floral buds of Cucumis sativus and fully sequenced. Their expression was studied by Northern analysis using two contrasting sex genotypes, an androecious line and a gynoecious one. The three genes are expressed at low levels at earlier bud stages, the levels rising as the bud matures. Two of the clones, CAG1 and CAG3, are expressed in the third and fourth whorl of mature flowers, while CAG2 is restricted to the carpel; none is expressed in leaves. The transcript levels do not appear to be modulated by gibberellin or ethephon, two treatments that alter sex expression in cucumber. While MADS-box genes probably play an essential role in cucumber floral development, as they do in other plants, our findings may imply that the pathway leading to reproductive organ arrest in cucumber unisexual buds acts independently of MADS-box gene expression.

title: Organ identity genes and modified patterns of flower development in Gerbera hybrida (Asteraceae)
abstract: We have used Gerbera hybrida (the cultivated ornamental, gerera) to investigate the molecular basis of flower development in Asteraceae, a family of flowering plants that have heteromorphic flowers and specialized floral organs. Flowers of the same genotype may differ in a number of parameters, including sex expression, symmetry, sympetaly and pigmentation. In order to study the role of organ identity determination in these phenomena we isolated and functionally analysed six MADS box genes from gerbera; these were shown by phylogenetic analysis to be orthologous to well characterized regulatory genes described from Arabidopsis and Antirrhinum. Expression analysis suggests that the two gerbera agamous orthologues, the globosa orthologue and one of the deficiens orthologues may have functional equivalency to their counterparts, participating in the C and B functions, respectively. However, the function of a second deficiens orthologue appears unrelated to the B function, and that of a squamosa orthologue seems distinct from squamosa as well as from the A function. The induction patterns of gerbera MADS box genes conform spatiotemporally to the multi-flowered, head-like inflorescence typical of Asteraceae. Furthermore, gerbera plants transgenic for the newly isolated MADS box genes shed light onto the mechanistic basis for some floral characteristics that are typical for Asteraceae. We can conclude, therefore, that the pappus bristles are sepals highly modified for seed dispersal, and that organ abortion in the female marginal flowers is dependent upon organ identity and not organ position when position is homeotically altered.

title: Floral Patterning in Lotus japonicus1[w]
abstract: Floral patterning in Papilionoideae plants, such as pea (Pisum sativum) and Medicago truncatula, is unique in terms of floral organ number, arrangement, and initiation timing as compared to other well-studied eudicots. To investigate the molecular mechanisms involved in the floral patterning in legumes, we have analyzed two mutants, proliferating floral meristem and proliferating floral organ-2 (pfo-2), obtained by ethyl methanesulfonate mutagenesis of Lotus japonicus. These two mutants showed similar phenotypes, with indeterminate floral structures and altered floral organ identities. We have demonstrated that loss of function of LjLFY and LjUFO/Pfo is likely to be responsible for these mutant phenotypes, respectively. To dissect the regulatory network controlling the floral patterning, we cloned homologs of the ABC function genes, which control floral organ identity in Arabidopsis (Arabidopsis thaliana). We found that some of the B and C function genes were duplicated. RNA in situ hybridization showed that the C function genes were expressed transiently in the carpel, continuously in stamens, and showed complementarity with the A function genes in the heterogeneous whorl. In proliferating floral meristem and pfo-2 mutants, all B function genes were down-regulated and the expression patterns of the A and C function genes were drastically altered. We conclude that LjLFY and LjUFO/Pfo are required for the activation of B function genes and function together in the recruitment and determination of petals and stamens. Our findings suggest that gene duplication, change in expression pattern, gain or loss of functional domains, and alteration of key gene functions all contribute to the divergence of floral patterning in L. japonicus.

title: Developmental specialisations in the legume family.
abstract: The legume family is astonishingly diverse; inventiveness in the form of novel organs, modified organs and additional meristems, is rife. Evolutionary changes can be inferred from the phylogenetic pattern of this diversity, but a full understanding of the origin of these 'hopeful monsters' of meristematic potential requires clear phylogenetic reconstructions and extensive, species-rich, sequence data. The task is large, but rapid progress is being made in both these areas. Here we review specialisations that have been characterised in a subset of intensively studied papilionoid legume taxa at the vanguard of developmental genetic studies.

title: A Change in SHATTERPROOF Protein Lies at the Origin of a Fruit Morphological Novelty and a New Strategy for Seed Dispersal in Medicago Genus1[C][W]
abstract: The evolutionary origin of a novel fruit morphology in legumes and the importance of changes in coding regions of master genes to generate diversity. Angiosperms are the most diverse and numerous group of plants, and it is generally accepted that this evolutionary success owes in part to the diversity found in fruits, key for protecting the developing seeds and ensuring seed dispersal. Although studies on the molecular basis of morphological innovations are few, they all illustrate the central role played by transcription factors acting as developmental regulators. Here, we show that a small change in the protein sequence of a MADS-box transcription factor correlates with the origin of a highly modified fruit morphology and the change in seed dispersal strategies that occurred in Medicago, a genus belonging to the large legume family. This protein sequence modification alters the functional properties of the protein, affecting the affinities for other protein partners involved in high-order complexes. Our work illustrates that variation in coding regions can generate evolutionary novelties not based on gene duplication/subfunctionalization but by interactions in complex networks, contributing also to the current debate on the relative importance of changes in regulatory or coding regions of master regulators in generating morphological novelties.

title: An efficient reverse genetics platform in the model legume Medicago truncatula.
abstract: Medicago truncatula is one of the model species for legume studies. In an effort to develop legume genetics resources, > 21 700 Tnt1 retrotransposon insertion lines have been generated. To facilitate fast-growing needs in functional genomics, two reverse genetics approaches have been established: web-based database searching and PCR-based reverse screening. More than 840 genes have been reverse screened using the PCR-based approach over the past 6 yr to identify mutants in these genes. Overall, c. 84% (705 genes) success rate was achieved in identifying mutants with at least one Tnt1 insertion, of which c. 50% (358 genes) had three or more alleles. To demonstrate the utility of the two reverse genetics platforms, two mutant alleles were isolated for each of the two floral homeotic MADS-box genes, MtPISTILATA and MtAGAMOUS. Molecular and genetic analyses indicate that Tnt1 insertions in exons of both genes are responsible for the defects in floral organ development. In summary, we have developed two efficient reverse genetics platforms to facilitate functional characterization of M. truncatula genes.

title: Insertional mutagenesis: a Swiss Army knife for functional genomics of Medicago truncatula.
abstract: Legumes are second only to grasses in worldwide economic importance, and understanding their molecular genetics is vital to the breeding of important grain and forage legumes. Over the past decade, Medicago truncatula has been selected as a model plant in which to study biological processes that are unique and pertinent to legumes, and that cannot easily be studied in Arabidopsis. Here, we discuss the most common tools for introducing and analyzing genetic mutations in M. truncatula. Because transformation and regeneration are still bottlenecks in studying a legume species, large-scale insertional mutagenesis poses a major challenge in M. truncatula. We discuss the tobacco retrotransposon Tnt1 as a viable and attractive option for introducing multiple independent insertions per plant for saturation mutagenesis.

title: Large-scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula.
abstract: Medicago truncatula is a fast-emerging model for the study of legume functional biology. We used the tobacco retrotransposon Tnt1 to tag the Medicago genome and generated over 7600 independent lines representing an estimated 190,000 insertion events. Tnt1 inserted on average at 25 different locations per genome during tissue culture, and insertions were stable during subsequent generations in soil. Analysis of 2461 Tnt1 flanking sequence tags (FSTs) revealed that Tnt1 appears to prefer gene-rich regions. The proportion of Tnt1 insertion in coding sequences was 34.1%, compared to the expected 15.9% if random insertions were to occur. However, Tnt1 showed neither unique target site specificity nor strong insertion hot spots, although some genes were more frequently tagged than others. Forward-genetic screening of 3237 R(1) lines resulted in identification of visible mutant phenotypes in approximately 30% of the regenerated lines. Tagging efficiency appears to be high, as all of the 20 mutants examined so far were found to be tagged. Taking the properties of Tnt1 into account and assuming 1.7 kb for the average M. truncatula gene size, we estimate that approximately 14,000-16,000 lines would be sufficient for 90% gene tagging coverage in M. truncatula. This is in contrast to more than 500,000 lines required to achieve the same saturation level using T-DNA tagging. Our data demonstrate that Tnt1 is an efficient insertional mutagen in M. truncatula, and could be a primary choice for other plant species with large genomes.

title: Reverse genetics in medicago truncatula using Tnt1 insertion mutants.
abstract: Medicago truncatula has been chosen as one of the two model species for legume molecular genetics and functional genomics studies. With the imminent completion of M. truncatula genome sequencing, availability of large-scale mutant populations becomes a priority. Over the last 5 years, nearly 12,000 insertion lines, which represent approximately 300,000 insertions, have been generated at the Samuel Roberts Noble Foundation using the tobacco retrotransposon Tnt1. Individual genomic DNA was isolated from each insertion line and pooled into four levels with the super-pool containing 500 lines. Using Tnt1-specific and gene-specific primers, a PCR-based efficient reverse screening strategy has been developed. Amplified PCR products are purified and sequenced to identify the exact insertion locations. Overall, approximately 90% of genes screened were found to have one or more Tnt1 insertions. Therefore, this PCR-based reverse screening is a rapid way of identifying knock-out mutants for specific genes in Tnt1-tagged population of M. truncatula. In addition to the DNA pool screening, a web-based database with more than 13,000 flanking sequence tags (FSTs) has also been set up. One can search the database to find an insertion line for the gene of interest.

title: Isolation of mtpim Proves Tnt1 a Useful Reverse Genetics Tool in Medicago truncatula and Uncovers New Aspects of AP1-Like Functions in Legumes1
abstract: Comparative studies help shed light on how the huge diversity in plant forms found in nature has been produced. We use legume species to study developmental differences in inflorescence architecture and flower ontogeny with classical models such as Arabidopsis thaliana or Antirrhinum majus. Whereas genetic control of these processes has been analyzed mostly in pea (Pisum sativum), Medicago truncatula is emerging as a promising alternative system for these studies due to the availability of a range of genetic tools. To assess the use of the retrotransposon Tnt1 for reverse genetics in M. truncatula, we screened a small Tnt1-mutagenized population using degenerate primers for MADS-box genes, known controllers of plant development. We describe here the characterization of mtpim, a new mutant caused by the insertion of Tnt1 in a homolog to the PROLIFERATING INFLORESCENCE MERISTEM (PIM)/APETALA1 (AP1)/SQUAMOSA genes. mtpim shows flower-to-inflorescence conversion and altered flowers with sepals transformed into leaves, indicating that MtPIM controls floral meristem identity and flower development. Although more extreme, this phenotype resembles the pea pim mutants, supporting the idea that M. truncatula could be used to complement analysis of reproductive development already initiated in pea. In fact, our study reveals aspects not shown by analysis of pea mutants: that the mutation in the AP1 homolog interferes with the specification of floral organs from common primordia and causes conversion of sepals into leaves, in addition to true conversion of flowers into inflorescences. The isolation of mtpim represents a proof of concept demonstrating that Tnt1 populations can be efficiently used in reverse genetics screenings in M. truncatula.

title: MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0.
abstract: We announce the release of the fourth version of MEGA software, which expands on the existing facilities for editing DNA sequence data from autosequencers, mining Web-databases, performing automatic and manual sequence alignment, analyzing sequence alignments to estimate evolutionary distances, inferring phylogenetic trees, and testing evolutionary hypotheses. Version 4 includes a unique facility to generate captions, written in figure legend format, in order to provide natural language descriptions of the models and methods used in the analyses. This facility aims to promote a better understanding of the underlying assumptions used in analyses, and of the results generated. Another new feature is the Maximum Composite Likelihood (MCL) method for estimating evolutionary distances between all pairs of sequences simultaneously, with and without incorporating rate variation among sites and substitution pattern heterogeneities among lineages. This MCL method also can be used to estimate transition/transversion bias and nucleotide substitution pattern without knowledge of the phylogenetic tree. This new version is a native 32-bit Windows application with multi-threading and multi-user supports, and it is also available to run in a Linux desktop environment (via the Wine compatibility layer) and on Intel-based Macintosh computers under the Parallels program. The current version of MEGA is available free of charge at (http://www.megasoftware.net).

title: A plant DNA minipreparation: Version II
abstract: The topic of this report is rap,d m,croscale methods for ,solat,on of plant D N A without tile use of ul tracentr ,fugatlon wEth CsCI. The D N A produced ,s of moderately high molecular weight and serves as a satisfactory substrate for most restrlctum cndonucleases and is statable for genom,c blot analys,s. In addi t ion to the rapidi ty and convenience of mlmpreps which permit a large number of samples to be processed in just a few hours, the small amount of tissue reqmred (less than 1.0 grams) allows tbr molecular analysis of plants at a very young stage Mm,prep D N A y,elds from leaf tissue of most species tested to date are typ,cally 30-100 big per gram tissue, greater than 50 kb, and remarkably uniform from sample to sample. The first mmlprep procedure we reported fi3r maize D N A isolation (Dellaporta et al , ;'*l,;tze Geneta3 Cr162162 Neu'_~letlrt. 1983) was adapted from a procedure commonly used for }'east D N A preparatmn (Dav,s et al. , 1980) Since th,s report, numerous personal commun,cat ,ons have demonstrated that the mm,prep procedure or a modification thereof, can be apphed to most plant species tested. For example, the method has been successfully used on Ntcottana hlgl~um. N. plumklgmgidtum. N. 3)/t'eJtrt~. L)s~opertcum sp.. Amar,mthm sp . Gl)~me max. Petuma h.~hra&. Several modifications have been apphed by these ,nvestlgators and in our own laboratory m order to extend the appl ,catmn of ram,prep procedures to other plant species. The select,on of a part icular protocol depends to a large degree on the plant spec,es used. However, the procedure reported here was selected to be statable for most situations.

title: Two O-linked N-acetylglucosamine transferase genes of Arabidopsis thaliana L. Heynh. have overlapping functions necessary for gamete and seed development.
abstract: The Arabidopsis SECRET AGENT (SEC) and SPINDLY (SPY) proteins are similar to animal O-linked N-acetylglucosamine transferases (OGTs). OGTs catalyze the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to Ser/Thr residues of proteins. In animals, O-GlcNAcylation has been shown to affect protein activity, stability, and/or localization. SEC protein expressed in Escherichia coli had autocatalytic OGT activity. To determine the function of SEC in plants, two tDNA insertional mutants were identified and analyzed. Although sec mutant plants did not exhibit obvious phenotypes, sec and spy mutations had a synthetic lethal interaction. This lethality was incompletely penetrant in gametes and completely penetrant postfertilization. The rate of both female and male sec spy gamete transmission was higher in plants heterozygous for both mutations than in plants heterozygous for sec and homozygous for spy. Double-mutant embryos aborted at various stages of development and no double-mutant seedlings were obtained. These results indicate that OGT activity is required during gametogenesis and embryogenesis with lethality occurring when parentally derived SEC, SPY, and/or O-GlcNAcylated proteins become limiting.

title: Virus-induced gene silencing as a tool for functional genomics in a legume species.
abstract: Virus-induced gene silencing (VIGS) is an attractive reverse-genetics tool for studies of gene function. However, efficient VIGS has only been accomplished in a few plant species. In order to extend the application of VIGS, we examined whether a VIGS vector based on Pea early browning virus (PEBV) would produce recognizable phenotypes in Pisum sativum. A plasmid vector of PEBV was modified to allow agro-inoculation and insertion of heterologous sequences. cDNA fragments of the P. sativum phytoene desaturase (PDS), LEAFY (LFY) and KORRIGAN1 (KOR1) homologues were inserted into the PEBV RNA2 vector, replacing the genes required for nematode transmission. Pisum sativum inoculated with PEBV carrying a fragment of PsPDS developed characteristic photo-bleached leaves and this phenotype was associated with a significant reduction in PsPDS mRNA. The P. sativum homologue of LFY is known as UNIFOLIATA (UNI). Plants inoculated with PEBV carrying a fragment of UNI developed distorted flowers and leaves with modified architecture, which are also observed in UNI-mutants. In Arabidopsis thaliana, the KOR1-mutant is characterized by an extreme dwarf phenotype. Pisum sativum plants inoculated with PEBV carrying a fragment of PsKOR1 displayed a significant reduction in height and inhibition of root growth. The PEBV VIGS vector did not affect the ability of P. sativum to flower, set seeds, and form nodules characteristic of symbiosis with rhizobium. These results suggest that the PEBV vector can be applied to functional genomics in a legume species to study genes involved in a wide range of biological processes.

title: Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.
abstract: The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities. Repeated application of Agrobacterium improved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.

title: Analysis of B function in legumes: PISTILLATA proteins do not require the PI motif for floral organ development in Medicago truncatula.
abstract: The B-class gene PISTILLATA (PI) codes for a MADS-box transcription factor required for floral organ identity in angiosperms. Unlike Arabidopsis, it has been suggested that legume PI genes contribute to a variety of processes, such as the development of floral organs, floral common petal-stamen primordia, complex leaves and N-fixing root nodules. Another interesting feature of legume PI homologues is that some of them lack the highly conserved C-terminal PI motif suggested to be crucial for function. Therefore, legume PI genes are useful for addressing controversial questions on the evolution of B-class gene function, including how they may have diverged in both function and structure to affect different developmental processes. However, functional analysis of legume PI genes has been hampered because no mutation in any B-class gene has been identified in legumes. Here we fill this gap by studying the PI function in the model legume species Medicago truncatula using mutant and RNAi approaches. Like other legume species, M. truncatula has two PI homologues. The expression of the two genes, MtPI and MtNGL9, has strongly diverged, suggesting differences in function. Our analyses show that these genes are required for petal and stamen identity, where MtPI appears to play a predominant role. However, they appear not to be required for development of the nodule, the common primordia or the complex leaf. Moreover, both M. truncatula PI homologues lack the PI motif, which indicates that the C-terminal motif is not essential for PI activity.

title: Functional specialization of duplicated AP3-like genes in Medicago truncatula.
abstract: The B-class of MADS box genes has been studied in a wide range of plant species, but has remained largely uncharacterized in legumes. Here we investigate the evolutionary fate of the duplicated AP3-like genes of a legume species. To obtain insight into the extent to which B-class MADS box gene functions are conserved or have diversified in legumes, we isolated and characterized the two members of the AP3 lineage in Medicago truncatula: MtNMH7 and MtTM6 (euAP3 and paleoAP3 genes, respectively). A non-overlapping and complementary expression pattern of both genes was observed in petals and stamens. MtTM6 was expressed predominantly in the outer cell layers of both floral organs, and MtNMH7 in the inner cell layers of petals and stamens. Functional analyses by reverse genetics approaches (RNAi and Tnt1 mutagenesis) showed that the contribution of MtNMH7 to petal identity is more important than that of MtTM6, whereas MtTM6 plays a more important role in stamen identity than its paralog MtNMH7. Our results suggest that the M. truncatula AP3-like genes have undergone a functional specialization process associated with complete partitioning of gene expression patterns of the ancestral gene lineage. We provide information regarding the similarities and differences in petal and stamen development among core eudicots.

title: Specific expression of the AGL1 MADS-box gene suggests regulatory functions in Arabidopsis gynoecium and ovule development.
abstract: Several members of the MADS-box gene family have been shown to be important regulators of flower development, controlling such well-studied early events as the formation of the floral meristem and the specification of floral organ identity. Other floral-specific MADS-box genes, of as yet unknown function, have been isolated by homology and are proposed to be part of a regulatory hierarchy controlling flower development. Some of these genes might regulate later aspects of flower development, such as development of individual floral organs, which is less well studied at the molecular level. This paper presents a detailed analysis of the expression pattern of one such gene from Arabidopsis, AGL1, using RNA in situ hybridization. It is found that AGL1 is specifically expressed in particular regions of the gynoecium and ovule, only during and after floral development stage 7. AGL1 expression at the tip of the growing carpel primordia, along the margins of the ovary valves in developing and mature gynoecia and in specific regions of developing and mature ovules provides important insights into the possible roles of AGL1. It is proposed that AGL1 may have regulatory functions in the structural definition and/or function of the valve margins, in axis maintenance during ovule development, in nutritional supply to the growing ovule and embryo sac, and in pollen tube guidance. In the floral homeotic mutants ag-1, ap3-3 and ap2-2, AGL1 mRNA is expressed in an organ-dependent manner, suggesting that AGL1 is a carpel-specific gene and as such ultimately depends upon the carpel identity gene AG for proper gene expression.

title: Prediction of Regulatory Interactions from Genome Sequences Using a Biophysical Model for the Arabidopsis LEAFY Transcription Factor[C][W]
abstract: This work presents the generation of a predictive model describing the DNA recognition specificity of the LEAFY floral transcription factor. The model is used to predict in vivo regulatory interactions between LEAFY and its target genes from mere inspection of various plant genome sequences. Despite great advances in sequencing technologies, generating functional information for nonmodel organisms remains a challenge. One solution lies in an improved ability to predict genetic circuits based on primary DNA sequence in combination with detailed knowledge of regulatory proteins that have been characterized in model species. Here, we focus on the LEAFY (LFY) transcription factor, a conserved master regulator of floral development. Starting with biochemical and structural information, we built a biophysical model describing LFY DNA binding specificity in vitro that accurately predicts in vivo LFY binding sites in the Arabidopsis thaliana genome. Applying the model to other plant species, we could follow the evolution of the regulatory relationship between LFY and the AGAMOUS (AG) subfamily of MADS box genes and show that this link predates the divergence between monocots and eudicots. Remarkably, our model succeeds in detecting the connection between LFY and AG homologs despite extensive variation in binding sites. This demonstrates that the cis-element fluidity recently observed in animals also exists in plants, but the challenges it poses can be overcome with predictions grounded in a biophysical model. Therefore, our work opens new avenues to deduce the structure of regulatory networks from mere inspection of genomic sequences.

title: Virus-induced gene silencing in Medicago truncatula and Lathyrus odorata.
abstract: Virus-induced gene silencing (VIGS) has become an important reverse genetics tool for functional genomics. VIGS vectors based on Pea early browning virus (PEBV, genus Tobravirus) and Bean pod mottle virus (genus Comovirus) are available for the legume species Pisum sativum and Glycine max, respectively. With the aim of extending the application of the PEBV VIGS vector to other legumes, we examined susceptibility of 99 accessions representing 24 legume species including 21 accessions of Medicago truncatula and 38 accessions Lotus japonicus. Infectivity of PEBV was tested by agro-inoculation with a vector carrying the complete beta-glucuronidase (GUS) coding sequence. In situ histochemical staining analysis indicated that 4 of 21 M. truncatula and three of three Lathyrus odorata accessions were infected systemically by GUS tagged PEBV, while none of 38 L. japonicus accessions displayed GUS staining of either inoculated or uninoculated leaves. Agro-inoculation of plants representing PEBV-GUS susceptible M. truncatula and L. odorata accessions with PEBV carrying a fragment of Phytoene desaturase (PDS) resulted in development of a bleaching phenotype suggesting a down-regulation of PDS expression. In M. truncatula this was supported by quantification of PDS mRNA levels by real-time PCR.

title: Manipulation of flower structure in transgenic tobacco
abstract: Genetic studies suggest that three homeotic functions, designated A, B, and C, act alone and together to specify the fate of floral organ primordia in distantly related dicotyledonous plant species. To test the genetic model, we have generated transgenic tobacco plants that ectopically express the AGAMOUS gene from Brassica napus, which is necessary for the C function. Flowers on the resulting plants showed homeotic transformations of sepals into carpels and petals into stamens. These phenotypes are consistent with predictions from the genetic model, show that expression of AGAMOUS is sufficient to provide ectopic C function, and demonstrate that the structure of flowers can be manipulated in a predictable manner by altering the expression of a single regulatory gene. Furthermore, the generation of the predicted transformations by ectopic expression of the Brassica gene in transgenic tobacco indicates that gene functions are interchangeable between phylogenetically distant species.

title: Ectopic expression of the floral homeotic gene AGAMOUS in transgenic Arabidopsis plants alters floral organ identity
abstract: The Arabidopsis floral homeotic gene AGAMOUS (AG) is required for development of the reproductive organs (stamens and carpels). In ag mutants, the loss of AG function leads to the conversion of these organs to the perianth organs (petals and sepals). In contrast, mutations in another floral homeotic gene, APETALA2 (AP2), result in the replacement of the perianth organs by the reproductive organs. On the basis of these observations, it has been proposed that AG and AP2 act in an antagonistic fashion. To test this hypothesis, we have studied the effects of ectopically expressed AG in transgenic Arabidopsis plants. The flowers of the transgenic plants exhibit a range of phenotypes mirroring those of ap2 mutants. These experiments provide direct evidence of the proposed antagonism between AG and AP2 functions, and the results strongly suggest that AG does indeed inhibit AP2 function.

title: Legume genome evolution viewed through the Medicago truncatula and Lotus japonicus genomes
abstract: Genome sequencing of the model legumes, Medicago truncatula and Lotus japonicus, provides an opportunity for large-scale sequence-based comparison of two genomes in the same plant family. Here we report synteny comparisons between these species, including details about chromosome relationships, large-scale synteny blocks, microsynteny within blocks, and genome regions lacking clear correspondence. The Lotus and Medicago genomes share a minimum of 10 large-scale synteny blocks, each with substantial collinearity and frequently extending the length of whole chromosome arms. The proportion of genes syntenic and collinear within each synteny block is relatively homogeneous. Medicago–Lotus comparisons also indicate similar and largely homogeneous gene densities, although gene-containing regions in Mt occupy 20–30% more space than Lj counterparts, primarily because of larger numbers of Mt retrotransposons. Because the interpretation of genome comparisons is complicated by large-scale genome duplications, we describe synteny, synonymous substitutions and phylogenetic analyses to identify and date a probable whole-genome duplication event. There is no direct evidence for any recent large-scale genome duplication in either Medicago or Lotus but instead a duplication predating speciation. Phylogenetic comparisons place this duplication within the Rosid I clade, clearly after the split between legumes and Salicaceae (poplar).

title: Genome-enabled insights into legume biology.
abstract: Legumes are the third-largest family of angiosperms, the second-most-important crop family, and a key source of biological nitrogen in agriculture. Recently, the genome sequences of Glycine max (soybean), Medicago truncatula, and Lotus japonicus were substantially completed. Comparisons among legume genomes reveal a key role for duplication, especially a whole-genome duplication event approximately 58 Mya that is shared by most agriculturally important legumes. A second and more recent genome duplication occurred only in the lineage leading to soybean. Outcomes of genome duplication, including gene fractionation and sub- and neofunctionalization, have played key roles in shaping legume genomes and in the evolution of legume-specific traits. Analysis of legume genome sequences also enables the discovery of legume-specific gene families and provides a framework for genome-wide association mapping that will target phenotypes of special importance in legumes. Translating genomic resources from sequenced species to less studied but still important "orphan" legumes will enhance prospects for world food production.

title: MADS-box genes in Ginkgo biloba and the evolution of the AGAMOUS family.
abstract: MADS-box proteins are a large family of transcription factors. In plants, many genes belonging to this family are involved in the homeosis of the floral system. Up to now, they have mainly been studied in angiosperms, especially in the model species Arabidopsis thaliana and Antirrhinum majus. We undertook a study of MADS-box genes in Ginkgo biloba, the unique extant representative of a whole branch of the phylogenetic tree of the seed plants. A polymerase chain reaction (PCR) survey reveals the diversity of MADS-box genes present in the genome of the Ginkgo. Duplications probably occurred specifically in the ginkgophyte lineage. Phylogenetic analyses revealed that one of these genes, GBM5, is an orthologue of the AGAMOUS gene of A. thaliana. We cloned and sequenced the entire cDNA of the GBM5 gene and studied its intron/exon structure. We showed by reverse transcriptase-PCR that it is expressed in both floral and vegetative tissues. We discuss the molecular evolution of the AGAMOUS family of genes.

title: MADS-box gene evolution-structure and transcription patterns.
abstract: This study presents a phylogenetic analysis of 198 MADS-box genes based on 420 parsimony-informative characters. The analysis includes only MIKC genes; therefore several genes from gymnosperms and pteridophytes are excluded. The strict consensus tree identifies all major monophyletic groups known from earlier analyses, and all major monophyletic groups are further supported by a common gene structure in exons 1-6 and by conserved C-terminal motifs. Transcription patterns are mapped on the tree to obtain an overview of MIKC gene transcription. Genes that are transcribed only in vegetative organs are located in the basal part of the tree, whereas genes involved in flower development have evolved later. As the universality of the ABC model has recently been questioned, special account is paid to the expression of A-, B-, and C-class genes. Mapping of transcription patterns on the phylogeny shows all three classes of MADS-box genes to be transcribed in the stamens and carpels. Thus the analysis does not support the ABC model as formulated at present.

title: Characterization of an AGAMOUS homologue from the conifer black spruce (Picea mariana) that produces floral homeotic conversions when expressed in Arabidopsis.
abstract: Advances in elucidating the molecular processes controlling flower initiation and development have provided unique opportunities to investigate the developmental genetics of non-flowering plants. In addition to providing insights into the evolutionary aspects of seed plants, identification of genes regulating reproductive organ development in gymnosperms could help determine the level of homology with current models of flower induction and floral organ identity. Based upon this, we have searched for putative developmental regulators in conifers with amino acid sequence homology to MADS-box genes. PCR cloning using degenerate primers targeted to the MADS-box domain revealed the presence of over 27 MADS-box genes within black spruce (Picea mariana), including several with extensive homology to either AP1 or AGAMOUS, both known to regulate flower development in Arabidopsis. This indicates that like angiosperms, conifers contain a large and diverse MADS-box gene family that probably includes regulators of reproductive organ development. Confirmation of this was provided by the characterization of an AGAMOUS-like cDNA clone called SAG1, whose conservation of intron position and tissue-specific expression within reproductive organs indicate that it is a homologue of AGAMOUS. Functional homology with AGAMOUS was demonstrated by the ability of SAG1 to produce homeotic conversions of sepals to carpels and petals to stamens when ectopically expressed in transgenic Arabidopsis. This suggests that some of the genetic pathways controlling flower and cone development are homologous, and antedate the 300-million-year-old divergence of angiosperms and gymnosperms.

title: A genetic framework for floral patterning
abstract: The initial steps of flower development involve two classes of consecutively acting regulatory genes. Meristem-identity genes, which act early to control the initiation of flowers, are expressed throughout the incipient floral primordium. Homeotic genes, which act later to specify the identity of individual floral organs, are expressed in distinct domains within the flower. The link between the two classes of genes has remained unknown so far. Here we show that the meristem-identity gene LEAFY has a role in controlling homeotic genes that is separable from its role in specifying floral fate. On the basis of our observation that LEAFY activates different homeotic genes through distinct mechanisms, we propose a genetic framework for the control of floral patterning.

title: Conserved intragenic elements were critical for the evolution of the floral C-function.
abstract: The floral C-function, which specifies stamen and carpel development, played a pivotal role in the evolution of flowers. An important aspect of this was the establishment of mechanisms regulating the temporal and spatial expression domain of the C-function genes. Transcription of the Arabidopsis C-function gene AGAMOUS (AG) is tightly controlled by factors that interact with cis-elements within its large second intron. Little is known about the regulatory role of intragenic elements in C-function genes from species other than Arabidopsis. We show that a binding site for the LEAFY (LFY) transcription factor, present in the AG intron, is conserved in the introns of diverse C-function genes and is positioned close to other conserved motifs. Using an in planta mutagenesis approach, we targeted evolutionarily conserved sequences in the intron of the Antirrhinum PLENA (PLE) gene to establish whether they regulate PLE expression. Small sequence deletions resulted in a novel class of heterochronic C-function mutants with delayed onset of PLE expression and loss of stamen identity. These phenotypes differ significantly from weak C-function mutant alleles in Antirrhinum and Arabidopsis. Our findings demonstrate that the PLE intron contains regulatory cis-elements, including a LFY-binding site, critical for establishing the correct C-function expression domain. We show that the LFY site, and other conserved intron elements, pre-date the divergence of the monocot and dicot lineages, suggesting that they were a determinant in the evolution of the C-function, and propose a threshold model to explain phenotypic divergence observed between C-function mutants.

title: Gene duplication and the evolution of plant MADS-box transcription factors.
abstract: Since the first MADS-box transcription factor genes were implicated in the establishment of floral organ identity in a couple of model plants, the size and scope of this gene family has begun to be appreciated in a much wider range of species. Over the course of millions of years the number of MADS-box genes in plants has increased to the point that the Arabidopsis genome contains more than 100. The understanding gained from studying the evolution, regulation and function of multiple MADS-box genes in an increasing set of species, makes this large plant transcription factor gene family an ideal subject to study the processes that lead to an increase in gene number and the selective birth, death and repurposing of its component members. Here we will use examples taken from the MADS-box gene family to review what is known about the factors that influence the loss and retention of genes duplicated in different ways and examine the varied fates of the retained genes and their associated biological outcomes.

title: Functional Analysis of the Arlequin Mutant Corroborates the Essential Role of the ARLEQUIN/TAGL1 Gene during Reproductive Development of Tomato
abstract: Reproductive development of higher plants comprises successive events of organ differentiation and growth which finally lead to the formation of a mature fruit. However, most of the genetic and molecular mechanisms which coordinate such developmental events are yet to be identified and characterized. Arlequin (Alq), a semi-dominant T-DNA tomato mutant showed developmental changes affecting flower and fruit ripening. Sepals were converted into fleshy organs which ripened as normal fruit organs and fruits displayed altered ripening features. Molecular characterization of the tagged gene demonstrated that it corresponded to the previously reported TOMATO AGAMOUS-LIKE 1 (TAGL1) gene, the tomato ortholog of SHATTERPROOF MADS-box genes of Arabidopsis thaliana, and that the Alq mutation promoted a gain-of-function phenotype caused by the ectopic expression of TAGL1. Ectopic overexpression of TAGL1 resulted in homeotic alterations affecting floral organ identity that were similar to but stronger than those observed in Alq mutant plants. Interestingly, TAGL1 RNAi plants yielded tomato fruits which were unable to ripen. They displayed a yellow-orange color and stiffness appearance which are in accordance with reduced lycopene and ethylene levels, respectively. Moreover, pericarp cells of TAGL1 RNAi fruits showed altered cellular and structural properties which correlated to both decreased expression of genes regulating cell division and lignin biosynthesis. Over-expression of TAGL1 is able to rescue the non-ripening phenotype of rin and nor mutants, which is mediated by the transcriptional activation of several ripening genes. Our results demonstrated that TAGL1 participates in the genetic control of flower and fruit development of tomato plants. Furthermore, gene silencing and over-expression experiments demonstrated that the fruit ripening process requires the regulatory activity of TAGL1. Therefore, TAGL1 could act as a linking factor connecting successive stages of reproductive development, from flower development to fruit maturation, allowing this complex process to be carried out successfully.

title: Multiple AGAMOUS Homologs from Cucumber and Petunia Differ in Their Ability to Induce Reproductive Organ Fate
abstract: The C function in Arabidopsis, which specifies stamen and carpel identity, is represented by a single gene called AGAMOUS (AG). From both petunia and cucumber, two MADS box genes have been isolated. Both share a high degree of amino acid sequence identity with the Arabidopsis AG protein. Their roles in specifying stamen and carpel identity have been studied by ectopic expression in petunia, resulting in plants with different floral phenotypes. Cucumber MADS box gene 1 (CUM1) induced severe homeotic transformations of sepals into carpelloid structures and petals into stamens, which is similar to ectopic AG expression in Arabidopsis plants. Overexpression of the other cucumber AG homolog, CUM10, resulted in plants with partial transformations of the petals into antheroid structures, indicating that CUM10 is also able to promote floral organ identity. From the two petunia AG homologs pMADS3 and Floral Binding Protein gene 6 (FBP6), only pMADS3 was able to induce homeotic transformations of sepals and petals. Ectopic expression of both pMADS3 and FBP6, as occurrs in the petunia homeotic mutant blind, phenocopies the pMADS3 single overexpresser plants, indicating that there is no additive effect of concerted expression. This study demonstrates that in petunia and cucumber, multiple AG homologs exist, although they differ in their ability to induce reproductive organ fate.

title: Ectopic expression of pMADS3 in transgenic petunia phenocopies the petunia blind mutant.
abstract: We cloned a MADS-box gene, pMADS3, from Petunia hybrida, which shows high sequence homology to the Arabidopsis AGAMOUS and Antirrhinum PLENA. pMADS3 is expressed exclusively in stamens and carpels of wild-type petunia plants. In the petunia mutant blind, which shows homeotic conversions of corolla limbs into antheroid structures with pollen grains and small parts of sepals into carpelloid tissue, pMADS3 is expressed in all floral organs as well as in leaves. Ectopic expression of pMADS3 in transgenic petunia leads to phenocopies of the blind mutant, i.e., the formation of antheroid structures on limbs and carpelloid tissue on sepals. Transgenic tobacco plants that overexpress pMADS3 exhibit an even more severe phenotype, with the sepals forming a carpel-like structure encasing the interior floral organs. Our results identify BLIND as a negative regulator of pMADS3, which specifies stamens and carpels during petunia flower development.

title: Single amino acid change alters the ability to specify male or female organ identity
abstract: The molecular mechanisms underlying the developmental processes that shape living organisms provide a basis to understand the evolution of biological complexity. Gene duplication allows biological functions to become separated, leading to increased complexity through subfunctionalization. Recently, the relative contributions to morphological evolution of changes to the regulatory and/or coding regions of duplicated genes have been the subject of debate. Duplication generated multiple copies of the MADS-box transcription factor genes that play essential roles in specifying organ identity in the flower, making this evolutionary novelty a good model to investigate the nature of the changes necessary to drive subfunctionalization. Here, we show that naturally occurring variation at a single amino acid in a MADS-box transcription factor switches its ability to specify male and female reproductive organs by altering its repertoire of protein–protein interactions. However, these different developmental fates are only manifest because of an underlying variation in the expression pattern of interacting proteins. This shows that the morphological outcomes of changes to protein sequence and gene expression must be interpreted in the context of the wider regulatory network. It also suggests an explanation for the surprisingly widespread duplications of some of the floral transcription factors.

title: Evolution in Action: Following Function in Duplicated Floral Homeotic Genes
abstract: Gene duplication plays a fundamental role in evolution by providing the genetic material from which novel functions can arise. Newly duplicated genes can be maintained by subfunctionalization (the duplicated genes perform different aspects of the original gene's function) and/or neofunctionalization (one of the genes acquires a novel function). PLENA in Antirrhinum and AGAMOUS in Arabidopsis are the canonical C-function genes that are essential for the specification of reproductive organs. These functionally equivalent genes encode closely related homeotic MADS-box transcription factors. Using genome synteny, we confirm phylogenetic analyses showing that PLENA and AGAMOUS are nonorthologous genes derived from a duplication in a common ancestor. Their respective orthologs, SHATTERPROOF in Arabidopsis and FARINELLI in Antirrhinum, have undergone independent subfunctionalization via changes in regulation and protein function. Surprisingly, the functional divergence between PLENA and FARINELLI, is morphologically manifest in both transgenic Antirrhinum and Arabidopsis. This provides a clear illustration of a random evolutionary trajectory for gene functions after a duplication event. Different members of a duplicated gene pair have retained the primary homeotic functions in different lineages, illustrating the role of chance in evolution. The differential ability of the Antirrhinum genes to promote male or female development provides a striking example of subfunctionalization at the protein level.

title: The Gene Balance Hypothesis: From Classical Genetics to Modern Genomics
abstract: The concept of genetic balance traces back to the early days of genetics. Additions or subtractions of single chromosomes to the karyotype (aneuploidy) produced greater impacts on the phenotype than whole-genome changes (ploidy). Studies on changes in gene expression in aneuploid and ploidy series

title: The gene balance hypothesis: implications for gene regulation, quantitative traits and evolution.
abstract: The gene balance hypothesis states that the stoichiometry of members of multisubunit complexes affects the function of the whole because of the kinetics and mode of assembly. Gene regulatory mechanisms also would be governed by these principles. Here, we review the impact of this concept with regard to the effects on the genetics of quantitative traits, the fate of duplication of genes following polyploidization events or segmental duplication, the basis of aneuploid syndromes, the constraints on cis and trans variation in gene regulation and the potential involvement in hybrid incompatibilities.

title: Gene and genome duplications: the impact of dosage-sensitivity on the fate of nuclear genes
abstract: Whole genome duplications (WGDs) followed by diploidization, which includes gene loss, have been an important recurrent process in the evolution of higher eukaryotes. Gene retention is biased to specific functional gene categories during diploidization. Dosage-sensitive genes, which include transcription factors, are significantly over-retained following WGDs. By contrast, these same functional gene categories exhibit lower retention rates following smaller scale duplications (e.g., local and tandem duplicates, segmental duplicates, aneuploidy). In light of these recent observations, we review current theories that address the fate of nuclear genes following duplication events (i.e., Gain of Function Hypothesis, Subfunctionalization Hypothesis, Increased Gene Dosage Hypothesis, Functional Buffering Model, and the Gene Balance Hypothesis). We broadly review different mechanisms of dosage-compensation that have evolved to alleviate harmful dosage-imbalances. In addition, we examine a recently proposed extension of the Gene Balance Hypothesis to explain the shared single copy status for a specific functional class of genes across the flowering plants. We speculate that the preferential retention of dosage-sensitive genes (e.g., regulatory genes such as transcription factors) and gene loss following WGDs has played a significant role in the development of morphological complexity in eukaryotes and facilitating speciation, respectively. Lastly, we will review recent findings that suggest polyploid lineages had increased rates of survival and speciation following mass extinction events, including the Cretaceous-Tertiary (KT) extinction.

title: Gene-balanced duplications, like tetraploidy, provide predictable drive to increase morphological complexity.
abstract: Controversy surrounds the apparent rising maximums of morphological complexity during eukaryotic evolution, with organisms increasing the number and nestedness of developmental areas as evidenced by morphological elaborations reflecting area boundaries. No "predictable drive" to increase this sort of complexity has been reported. Recent genetic data and theory in the general area of gene dosage effects has engendered a robust "gene balance hypothesis," with a theoretical base that makes specific predictions as to gene content changes following different types of gene duplication. Genomic data from both chordate and angiosperm genomes fit these predictions: Each type of duplication provides a one-way injection of a biased set of genes into the gene pool. Tetraploidies and balanced segments inject bias for those genes whose products are the subunits of the most complex biological machines or cascades, like transcription factors (TFs) and proteasome core proteins. Most duplicate genes are removed after tetraploidy. Genic balance is maintained by not removing those genes that are dose-sensitive, which tends to leave duplicate "functional modules" as the indirect products (spandrels) of purifying selection. Functional modules are the likely precursors of coadapted gene complexes, a unit of natural selection. The result is a predictable drive mechanism where "drive" is used rigorously, as in "meiotic drive." Rising morphological gain is expected given a supply of duplicate functional modules. All flowering plants have survived at least three large-scale duplications/diploidizations over the last 300 million years (Myr). An equivalent period of tetraploidy and body plan evolution may have ended for animals 500 million years ago (Mya). We argue that "balanced gene drive" is a sufficient explanation for the trend that the maximums of morphological complexity have gone up, and not down, in both plant and animal eukaryotic lineages.