Genome-wide identification and characterization of MADS-box family genes related to organ development and stress resistance in Brassica rapa

BackgroundMADS-box transcription factors (TFs) are important in floral organ specification as well as several other aspects of plant growth and development. Studies on stress resistance-related functions of MADS-box genes are very limited and no such functional studies in Brassica rapa have been reported. To gain insight into this gene family and to elucidate their roles in organ development and stress resistance, we performed genome-wide identification, characterization and expression analysis of MADS-box genes in B. rapa.ResultsWhole-genome survey of B. rapa revealed 167 MADS-box genes, which were categorized into type I (Mα, Mβ and Mγ) and type II (MIKCc and MIKC*) based on phylogeny, protein motif structure and exon-intron organization. Expression analysis of 89 MIKCc and 11 MIKC* genes was then carried out. In addition to those with floral and vegetative tissue expression, we identified MADS-box genes with constitutive expression patterns at different stages of flower development. More importantly, from a low temperature-treated whole-genome microarray data set, 19 BrMADS genes were found to show variable transcript abundance in two contrasting inbred lines of B. rapa. Among these, 13 BrMADS genes were further validated and their differential expression was monitored in response to cold stress in the same two lines via qPCR expression analysis. Additionally, the set of 19 BrMADS genes was analyzed under drought and salt stress, and 8 and 6 genes were found to be induced by drought and salt, respectively.ConclusionThe extensive annotation and transcriptome profiling reported in this study will be useful for understanding the involvement of MADS-box genes in stress resistance in addition to their growth and developmental functions, which ultimately provides the basis for functional characterization and exploitation of the candidate genes for genetic engineering of B. rapa.

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title: MADS-box gene evolution beyond flowers: expression in pollen, endosperm, guard cells, roots and trichomes.
abstract: MADS-box genes encode transcriptional regulators involved in diverse aspects of plant development. Here we describe the cloning and mRNA spatio-temporal expression patterns of five new MADS-box genes from Arabidopsis: AGL16, AGL18, AGL19, AGL27 and AGL31. These genes will probably become important molecular tools for both evolutionary and functional analyses of vegetative structures. We mapped our data and previous expression patterns onto a new MADS-box phylogeny. These analyses suggest that the evolution of the MADS-box family has involved a rapid and simultaneous functional diversification in vegetative as well as reproductive structures. The hypothetical ancestral genes had broader expression patterns than more derived ones, which have been co-opted for putative specialized functions as suggested by their expression patterns. AGL27 and AGL31, which are closely related to the recently described flowering-time gene FLC (previously AGL25), are expressed in most plant tissues. AGL19 is specifically expressed in the outer layers of the root meristem (lateral root cap and epidermis) and in the central cylinder cells of mature roots. AGL18, which is most similar in sequence to the embryo-expressed AGL15 gene, is expressed in the endosperm and in developing male and female gametophytes, suggesting a role for AGL18 that is distinct from previously characterized MADS-box genes. Finally, AGL16 RNA accumulates in leaf guard cells and trichomes. Our new phylogeny reveals seven new monophyletic clades of MADS-box sequences not specific to flowers, suggesting that complex regulatory networks involving several MADS-box genes, similar to those that control flower development, underlie development of vegetative structures.

title: Role of MADS box proteins and their cofactors in combinatorial control of gene expression and cell development.
abstract: In all organisms, correct development, growth and function depends on the precise and integrated control of the expression of their genes. Often, gene regulation depends upon the cooperative binding of proteins to DNA and upon protein-protein interactions. Eukaryotes have widely exploited combinatorial strategies to create gene regulatory networks. MADS box proteins constitute the perfect example of cellular coordinators. These proteins belong to a large family of transcription factors present in most eukaryotic organisms and are involved in diverse and important biological functions. MADS box proteins are combinatorial transcription factors in that they often derive their regulatory specificity from other DNA binding or accessory factors. This review is aimed at analyzing how MADS box proteins combine with a variety of cofactors to achieve functional diversity.

title: trans meets cis in MADS science.
abstract: The interaction between a transcription factor and its binding site at the DNA is an integral part of transcriptional regulatory networks, which is fundamental for an understanding of biological processes. An example is the family of MADS domain transcription factors, which represent key regulators of processes in yeast, animals and plants. However, despite our extensive knowledge of these transcription factors, limited information is available on the cis-elements to which these proteins bind or how these elements are defined. Here, we discuss the current understanding of MADS protein binding sites and compare data from various organisms. This information can help us in developing algorithms to predict binding sites for MADS domain transcription factors, which would be a significant step forward in the identification of "down-stream" target genes and the elucidation of transcriptional networks.

title: The unfolding drama of flower development: recent results from genetic and molecular analyses.
abstract: There has been an explosion of information about flower development recently, largely because of genetic and molecular studies in Arabidopsis thaliana and Antirrhihum majus. A number of homeotic genes have been identified that regulate flower development, and models have been proposed for the specification of meristem and floral organ identities. Molecular cloning of many of these genes has allowed the testing of specific predictions of the models but also has led to modifications of a floral organ identity model. Furthermore, several of the floral genes contain a conserved region, the MADS box, which encodes a domain with striking sequence similarity with known transcription factors from human and yeast. Additional MADS box genes have been isolated from several plants; these genes are likely to play important regulatory roles during flower development. The genetic and molecular studies have uncovered many of the components of a complex network of regulatory proteins that directs flower development. Further characterization of these and other yet to be discovered components promises to contribute a great deal to our understanding of the mechanisms controlling flower development. Flowering plants, like other land plants, have vegetative organs such as roots, stems, and leaves, which absorb nutrients and water from the soil, transport them to other parts of the plant, and synthesize organic compounds using the sun's energy. In addition, flowering plants produce elaborate reproductive structures, the flowers, which, following fertilization, become fruits and bear seeds. From the seasoned gardener to the casual observer, from the naturalist to the florist shopper, people have always been fascinated by the enormous variety of flowers, ranging from 2 mm to > 10 cm in length, covering the whole visual spectrum with their colors, and differing in the arrangement of flowers and the symmetry within a flower. How do flowers develop? What genes regulate this complex process? In recent years, rapid advances have been made in addressing these questions, largely as a result of genetic and molecular studies in two distantly related flowering plants, Arabidopsis thaliana, a relative of cauliflower and cabbage, and the snapdragon, Antirrhinum majus.

title: A MADS-Box Gene Necessary for Fruit Ripening at the Tomato Ripening-Inhibitor (Rin) Locus
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title: Isolation and properties of cDNA clones encoding SRF, a transcription factor that binds to the c-fos serum response element
abstract: The serum response element (SRE) is a sequence required for transient transcriptional activation of genes in response to growth factors. We have isolated cDNA clones encoding serum response factor (SRF), a ubiquitous nuclear protein that binds to the SRE. The SRF gene is highly conserved through evolution, and in cultured cells its transcription is itself transiently increased following serum stimulation. A cDNA clone of SRF expressed in vitro generates protein that forms complexes indistinguishable from those formed with HeLa cell SRF, as judged by DNA binding specificity and the ability to promote SRE-dependent in vitro transcription. SRF binds DNA as a dimer, and the DNA binding/dimerization domain of the protein exhibits striking homology to two yeast regulatory proteins.

title: An ancestral MADS-box gene duplication occurred before the divergence of plants and animals.
abstract: Changes in genes encoding transcriptional regulators can alter development and are important components of the molecular mechanisms of morphological evolution. MADS-box genes encode transcriptional regulators of diverse and important biological functions. In plants, MADS-box genes regulate flower, fruit, leaf, and root development. Recent sequencing efforts in Arabidopsis have allowed a nearly complete sampling of the MADS-box gene family from a single plant, something that was lacking in previous phylogenetic studies. To test the long-suspected parallel between the evolution of the MADS-box gene family and the evolution of plant form, a polarized gene phylogeny is necessary. Here we suggest that a gene duplication ancestral to the divergence of plants and animals gave rise to two main lineages of MADS-box genes: TypeI and TypeII. We locate the root of the eukaryotic MADS-box gene family between these two lineages. A novel monophyletic group of plant MADS domains (AGL34 like) seems to be more closely related to previously identified animal SRF-like MADS domains to form TypeI lineage. Most other plant sequences form a clear monophyletic group with animal MEF2-like domains to form TypeII lineage. Only plant TypeII members have a K domain that is downstream of the MADS domain in most plant members previously identified. This suggests that the K domain evolved after the duplication that gave rise to the two lineages. Finally, a group of intermediate plant sequences could be the result of recombination events. These analyses may guide the search for MADS-box sequences in basal eukaryotes and the phylogenetic placement of new genes from other plant species.

title: Evolution and divergence of the MADS-box gene family based on genome-wide expression analyses.
abstract: MADS-box genes encode transcription factors involved in various important aspects of development and differentiation in land plants, metazoans, and other organisms. Three types of land plant MADS-box genes have been reported. MIKCC- and MIKC*-type genes both contain conserved MADS and K domains but have different exon/intron structures. M-type genes lack a K domain. Most MADS-box genes previously analyzed in land plants are expressed in the sporophyte (diploid plant body); few are expressed in the gametophyte (haploid plant body). Land plants are believed to have evolved from a gametophyte (haploid)-dominant ancestor without a multicellular sporophyte (diploid plant body); most genes expressed in the sporophyte probably originated from those used in the gametophyte during the evolution of land plants. To analyze the evolution and diversification of MADS-box genes in land plants, gametophytic MADS-box genes were screened using macroarray analyses for 105 MADS-box genes found in the Arabidopsis genome. Eight MADS-box genes were predominantly expressed in pollen, the male gametophyte; all but one of their expression patterns was confirmed by Northern analyses. Analyses of the exon/intron structure of these seven genes revealed that they included two MIKCC-type, one M-type, and four MIKC*-type MADS-box genes. Previously, MIKC*-type genes have been reported only from a moss and a club moss, and this is the first record in seed plants. These genes can be used to investigate the unknown ancestral functions of MADS-box genes in land plants. The macroarray analyses did not detect expression of 56 of 61 M-type MADS-box genes in any tissues examined. A phylogenetic tree including all three types of Arabidopsis MADS-box genes with representative genes from other organisms showed that M-type genes were polyphyletic and that their branch lengths were much longer than for the other genes. This finding suggests that most M-type genes are pseudogenes, although further experiments are necessary to confirm this possibility. Our global phylogenetic analyses of MADS-box genes did not support the previous classification of MADS-box genes into type I and II groups, based on smaller scale analyses. An evolutionary scenario for the evolution of MADS-box genes in land plants is discussed.

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: Classification and phylogeny of the MADS-box multigene family suggest defined roles of MADS-box gene subfamilies in the morphological evolution of eukaryotes
abstract: The MADS-box encodes a novel type of DNA-binding domain found so far in a diverse group of transcription factors from yeast, animals, and seed plants. Here, our first aim was to evaluate the primary structure of the MADS-box. Compilation of the 107 currently available MADS-domain sequences resulted in a signature which can strictly discriminate between genes possessing or lacking a MADS-domain and allowed a classification of MADS-domain proteins into several distinct subfamilies. A comprehensive phylogenetic analysis of known eukaryotic MADS-box genes, which is the first comprising animal as well as fungal and plant homologs, showed that the vast majority of subfamily members appear on distinct subtrees of phylogenetic trees, suggesting that subfamilies represent monophyletic gene clades and providing the proposed classification scheme with a sound evolutionary basis. A reconstruction of the history of the MADS-box gene subfamilies based on the taxonomic distribution of contemporary subfamily members revealed that each subfamily comprises highly conserved putative orthologs and recent paralogs. Some subfamilies must be very old (1,000 MY or more), while others are more recent. In general, subfamily members tend to share highly similar sequences, expression patterns, and related functions. The defined species distribution, specific function, and strong evolutionary conservation of the members of most subfamilies suggest that the establishment of different subfamilies was followed by rapid fixation and was thus highly advantageous during eukaryotic evolution. These gene subfamilies may have been essential prerequisites for the establishment of several complex eukaryotic body structures, such as muscles in animals and certain reproductive structures in higher plants, and of some signal transduction pathways. Phylogenetic trees indicate that after establishment of different subfamilies, additional gene duplications led to a further increase in the number of MADS-box genes. However, several molecular mechanisms of MADS-box gene diversification were used to a quite different extent during animal and plant evolution. Known plant MADS-domain sequences diverged much faster than those of animals, and gene duplication and sequence diversification were extensively used for the creation of new genes during plant evolution, resulting in a relatively large number of interacting genes. In contrast, the available data on animal genes suggest that increase in gene number was only moderate in the lineage leading to mammals, but in the case of MEF2-like gene products, heterodimerization between different splice variants may have increased the combinatorial possibilities of interactions considerably. These observations demonstrate that in metazoan and plant evolution, increased combinatorial possibilities of MADS-box gene product interactions correlated with the evolution of increasingly complex body plans.

title: The K domain mediates heterodimerization of the Arabidopsis floral organ identity proteins, APETALA3 and PISTILLATA.
abstract: MADS genes in plants encode key developmental regulators of vegetative and reproductive development. The majority of well-characterized plant MADS proteins contain two conserved domains, the DNA-binding MADS domain and the K domain. The K domain is predicted to form three amphipathic alpha-helices referred to as K1, K2, and K3. In this report, we define amino acids and subdomains important for heterodimerization between the two Arabidopsis floral organ identity MADS proteins APETALA3 (AP3) and PISTILLATA (PI). Analysis of mutants defective in dimerization demonstrates that K1, K2 and the region between K1 and K2 are critical for the strength of AP3/PI dimerization. The majority of the critical amino acids are hydrophobic indicating that the K domain mediates AP3/PI interaction primarily through hydrophobic interactions. Specially, K1 of AP3 and PI resembles a leucine zipper motif. Most mutants defective in AP3/PI heterodimerization in yeast exhibit partial floral organ identity function in transgenic Arabidopsis. Our results also indicate that the motif containing Asn-98 and specific charged residues in K1 (Glu-97 in PI and Arg-102 in AP3) are important for both the strength and specificity of AP3/PI heterodimer formation.

title: Two ancient classes of MIKC-type MADS-box genes are present in the moss Physcomitrella patens.
abstract: Characterization of seven MADS-box genes, termed PPM1-PPM4 and PpMADS1-PpMADS3, from the moss model species Physcomitrella patens is reported. Phylogeny reconstructions and comparison of exon-intron structures revealed that the genes described here represent two different classes of homologous, yet distinct, MIKC-type MADS-box genes, termed MIKC(c)-type genes-"(c)" stands for "classic"-(PPM1, PPM2, PpMADS1) and MIKC(*)-type genes (PPM3, PPM4, PpMADS2, PpMADS3). The two gene classes deviate from each other in a characteristic way, especially in a sequence stretch termed intervening region. MIKC(c)-type genes are abundantly present in all land plants which have been investigated in this respect, and give rise to well-known gene types such as floral meristem and organ identity genes. In contrast, LAMB1 from the clubmoss Lycopodium annotinum was identified as the only other MIKC(*)-type gene published so far. Our findings strongly suggest that the most recent common ancestor of mosses and vascular plants contained at least one MIKC(c)-type and one MIKC(*)-type gene. Our studies thus reveal an ancient duplication of an MIKC-type gene that occurred before the separation of the lineages that led to extant mosses and vascular plants more than about 450 MYA. The identification of bona fide K-domains in both MIKC(*)-type and MIKC(c)-type proteins suggests that the K-domain is more ancient than is suggested by a recent alternative hypothesis. MIKC(*)-type genes may have escaped identification in ferns and seed plants so far. It seems more likely, however, that they represent a class of genes which has been lost in the lineage which led to extant ferns and seed plants. The high number of P. patens MADS-box genes and the presence of a K-box in the coding region and of some potential binding sites for MADS-domain proteins and other transcription factors in the putative promoter regions of these genes suggest that MADS-box genes in mosses are involved in complex gene regulatory networks similar to those in flowering plants.

title: Complexes of MADS-box proteins are sufficient to convert leaves into floral organs
abstract: Genetic studies, using floral homeotic mutants, have led to the ABC model of flower development. This model proposes that the combinatorial action of three sets of genes, the A, B and C function genes, specify the four floral organs (sepals, petals, stamens and carpels) in the concentric floral whorls. However, attempts to convert vegetative organs into floral organs by altering the expression of ABC genes have been unsuccessful. Here we show that the class B proteins of Arabidopsis, PISTILLATA (PI) and APETALA3 (AP3), interact with APETALA1 (AP1, a class A protein) and SEPALLATA3 (SEP3, previously AGL9), and with AGAMOUS (AG, a class C protein) through SEP3. We also show that vegetative leaves of triply transgenic plants, 35S::PI;35S::AP3;35S::AP1 or 35S::PI;35S::AP3;35S::SEP3, are transformed into petaloid organs and that those of 35S::PI; 35S::AP3;35S::SEP3;35S::AG are transformed into staminoid organs. Our findings indicate that the formation of ternary and quaternary complexes of ABC proteins may be the molecular basis of the ABC model, and that the flower-specific expression of SEP3 restricts the action of the ABC genes to the flower.

title: Diverse roles for MADS box genes in Arabidopsis development.
abstract: Members of the MADS box gene family play important roles in flower development from the early step of determining the identity of floral meristems to specifying the identity of floral organ primordia later in flower development. We describe here the isolation and characterization of six additional members of this family, increasing the number of reported Arabidopsis MADS box genes to 17. All 11 members reported prior to this study are expressed in flowers, and the majority of them are floral specific. RNA expression analyses of the six genes reported here indicate that two genes, AGL11 and AGL13 (AGL for AGAMOUS-like), are preferentially expressed in ovules, but each has a distinct expression pattern. AGL15 is preferentially expressed in embryos, with its onset at or before the octant stage early in embryo development. AGL12, AGL14, and AGL17 are all preferentially expressed in root tissues and therefore represent the only characterized MADS box genes expressed in roots. Phylogenetic analyses showed that the two genes expressed in ovules are closely related to previously isolated MADS box genes, whereas the four genes showing nonfloral expression are more distantly related. Data from this and previous studies indicate that in addition to their proven role in flower development, MADS box genes are likely to play roles in many other aspects of plant development.

title: MADS-box genes are involved in floral development and evolution.
abstract: MADS-box genes encode transcription factors in all eukaryotic organisms thus far studied. Plant MADS-box proteins contain a DNA-binding (M), an intervening (I), a Keratin-like (K) and a C-terminal C-domain, thus plant MADS-box proteins are of the MIKC type. In higher plants most of the well-characterized genes are involved in floral development. They control the transition from vegetative to generative growth and determine inflorescence meristem identity. They specify floral organ identity as outlined in the ABC model of floral development. Moreover, in Antirrhinum majus the MADS-box gene products DEF/GLO and PLE control cell proliferation in the developing flower bud. In this species the DEF/GLO and the SQUA proteins form a ternary complex which determines the overall "Bauplan" of the flower. Phylogenetic reconstructions of MADS-box sequences obtained from ferns, gymnosperms and higher eudicots reveal that, although ferns possess already MIKC type genes, these are not orthologous to the well characterized MADS-box genes from gymnosperms or angiosperms. Putative orthologs of floral homeotic B- and C-function genes have been identified in different gymnosperms suggesting that these genes evolved some 300-400 million years ago. Both gymnosperms and angiosperms also contain a hitherto unknown sister clade of the B-genes, which we termed Bsister. A novel hypothesis will be described suggesting that B and Bsister might be involved in sex determination of male and female reproductive organs, respectively.

title: AGL24 acts as a promoter of flowering in Arabidopsis and is positively regulated by vernalization.
abstract: MADS-domain-containing transcription factors comprise a large family of regulators that have diverse roles in plant development, including the regulation of flowering time. AGAMOUS-LIKE 20/SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) and FRUITFUL act to promote flowering, whereas FLOWERING LOCUS C (FLC), FLOWERING LOCUS M/MADS AFFECTING FLOWERING1, and SHORT VEGETATIVE PHASE are inhibitors of flowering. Here we report that AGAMOUS-LIKE 24 (AGL24) also plays a role in the regulation of flowering time. agl24 mutants are late flowering and overexpression of AGL24 causes early flowering in wild-type and late-flowering-mutant backgrounds. The effect of AGL24 overexpression is most pronounced in autonomous-pathway-mutant and FRIGIDA-containing backgrounds. The behavior of AGL24 is most similar to that of SOC1. Like SOC1, AGL24 mRNA levels are upregulated by vernalization. Unlike SOC1, however, AGL24 mRNA levels are not affected by FLC, and therefore AGL24 may represent an FLC-independent target of the vernalization pathway. There is also evidence for cross-talk between AGL24 and SOC1. When overexpressed, SOC1 and AGL24 are able to upregulate each other's expression. Thus, AGL24 represents another component in a network of MADS-domain-containing transcription factors that regulate flowering time.

title: Redundant regulation of meristem identity and plant architecture by FRUITFULL, APETALA1 and CAULIFLOWER.
abstract: The transition from vegetative to reproductive phases during Arabidopsis development is the result of a complex interaction of environmental and endogenous factors. One of the key regulators of this transition is LEAFY (LFY), whose threshold levels of activity are proposed to mediate the initiation of flowers. The closely related APETALA1 (AP1) and CAULIFLOWER (CAL) meristem identity genes are also important for flower initiation, in part because of their roles in upregulating LFY expression. We have found that mutations in the FRUITFULL (FUL) MADS-box gene, when combined with mutations in AP1 and CAL, lead to a dramatic non-flowering phenotype in which plants continuously elaborate leafy shoots in place of flowers. We demonstrate that this phenotype is caused both by the lack of LFY upregulation and by the ectopic expression of the TERMINAL FLOWER1 (TFL1) gene. Our results suggest that the FUL, AP1 and CAL genes act redundantly to control inflorescence architecture by affecting the domains of LFY and TFL1 expression as well as the relative levels of their activities.

title: B and C floral organ identity functions require SEPALLATA MADS-box genes
abstract: Abnormal flowers have been recognized for thousands of years, but only in the past decade have the mysteries of flower development begun to unfold. Among these mysteries is the differentiation of four distinct organ types (sepals, petals, stamens and carpels), each of which may be a modified leaf. A landmark accomplishment in plant developmental biology is the ABC model of flower organ identity. This simple model provides a conceptual framework for explaining how the individual and combined activities of the ABC genes produce the four organ types of the typical eudicot flower. Here we show that the activities of the B and C organ-identity genes require the activities of three closely related and functionally redundant MADS-box genes, SEPALLATA1/2/3 (SEP1/2/3). Triple mutant Arabidopsis plants lacking the activity of all three SEP genes produce flowers in which all organs develop as sepals. Thus SEP1/2/3 are a class of organ-identity genes that is required for development of petals, stamens and carpels.

title: The FRUITFULL MADS-box gene mediates cell differentiation during Arabidopsis fruit development.
abstract: Fruit morphogenesis is a process unique to flowering plants, and yet little is known about its developmental control. Following fertilization, fruits typically undergo a dramatic enlargement that is accompanied by differentiation of numerous distinct cell types. We have identified a mutation in Arabidopsis called fruitfull (ful-1), which abolishes elongation of the silique after fertilization. The ful-1 mutation is caused by the insertion of a DsE transposable enhancer trap element into the 5' untranslated leader of the AGL8 MADS-box gene. beta-glucuronidase (GUS) reporter gene expression in the enhancer trap line is observed specifically in all cell layers of the valve tissue, but not in the replum, the septum or the seeds, and faithfully mimics RNA in situ hybridization data reported previously. The lack of coordinated growth of the fruit tissues leads to crowded seeds, a failure of dehiscence and, frequently, the premature rupture of the carpel valves. The primary defect of ful-1 fruits is within the valves, whose cells fail to elongate and differentiate. Stomata, which are frequent along the epidermis of wild-type valves, are completely eliminated in the ful mutant valves. In addition to the effect on fruit development, ful cauline leaves are broader than those of wild type and show a reduction in the number of internal cell layers. These data suggest that AGL8/FUL regulates the transcription of genes required for cellular differentiation during fruit and leaf development.

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: The TRANSPARENT TESTA16 Locus Encodes the ARABIDOPSIS BSISTER MADS Domain Protein and Is Required for Proper Development and Pigmentation of the Seed Coat Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.004127.
abstract: Screening for seed pigmentation phenotypes in Arabidopsis led to the isolation of three allelic yellow-seeded mutants, which defined the novel TRANSPARENT TESTA16 (TT16) locus. Cloning of TT16 was performed by T-DNA tagging and confirmed by genetic complementation and sequencing of two mutant alleles. TT16 encodes the ARABIDOPSIS BSISTER (ABS) MADS domain protein. ABS belongs to the recently identified “B-sister” (BS) clade, which contains genes of unknown function that are expressed mainly in female organs. Phylogenetic analyses using a maximum parsimony approach confirmed that TT16/ABS and related proteins form a monophyletic group. TT16/ABS was expressed mainly in the ovule, as are the other members of the BS clade. TT16/ABS is necessary for BANYULS expression and proanthocyanidin accumulation in the endothelium of the seed coat, with the exception of the chalazal-micropylar area. In addition, mutant phenotype and ectopic expression analyses suggested that TT16/ABS also is involved in the specification of endothelial cells. Nevertheless, TT16/ABS apparently is not required for proper ovule function. We report the functional characterization of a member of the BS MADS box gene subfamily, demonstrating its involvement in endothelial cell specification as well as in the increasingly complex genetic control of flavonoid biosynthesis in the Arabidopsis seed coat.

title: The SEP4 Gene of Arabidopsis thaliana Functions in Floral Organ and Meristem Identity
abstract: The ABC model of flower organ identity is widely recognized as providing a framework for understanding the specification of flower organs in diverse plant species. Recent studies in Arabidopsis thaliana have shown that three closely related MADS-box genes, SEPALLATA1 (SEP1), SEP2 and SEP3, are required to specify petals, stamens, and carpels because these organs are converted into sepals in sep1 sep2 sep3 triple mutants. Additional studies indicate that the SEP proteins form multimeric complexes with the products of the B and C organ identity genes. Here, we characterize the SEP4 gene, which shares extensive sequence similarity to and an overlapping expression pattern with the other SEP genes. Although sep4 single mutants display a phenotype similar to that of wild-type plants, we find that floral organs are converted into leaf-like organs in sep1 sep2 sep3 sep4 quadruple mutants, indicating the involvement of all four SEP genes in the development of sepals. We also find that SEP4 contributes to the development of petals, stamens, and carpels in addition to sepals and that it plays an important role in meristem identity. These and other data demonstrate that the SEP genes play central roles in flower meristem identity and organ identity.

title: Molecular mechanisms underlying origin and diversification of the angiosperm flower.
abstract: Understanding the mode and mechanisms of the evolution of the angiosperm flower is a long-standing and central problem of evolutionary biology and botany. It has essentially remained unsolved, however. In contrast, considerable progress has recently been made in our understanding of the genetic basis of flower development in some extant model species. The knowledge that accumulated this way has been pulled together in two major hypotheses, termed the 'ABC model' and the 'floral quartet model'. These models explain how the identity of the different types of floral organs is specified during flower development by homeotic selector genes encoding transcription factors.



We intend to explain how the 'ABC model' and the 'floral quartet model' are now guiding investigations that help to understand the origin and diversification of the angiosperm flower.



Investigation of orthologues of class B and class C floral homeotic genes in gymnosperms suggest that bisexuality was one of the first innovations during the origin of the flower. The transition from dimer to tetramer formation of floral homeotic proteins after establishment of class E proteins may have increased cooperativity of DNA binding of the transcription factors controlling reproductive growth. That way, we hypothesize, better 'developmental switches' originated that facilitated the early evolution of the flower. Expression studies of ABC genes in basally diverging angiosperm lineages, monocots and basal eudicots suggest that the 'classical' ABC system known from core eudicots originated from a more fuzzy system with fading borders of gene expression and gradual transitions in organ identity, by sharpening of ABC gene expression domains and organ borders. Shifting boundaries of ABC gene expression may have contributed to the diversification of the angiosperm flower many times independently, as may have changes in interactions between ABC genes and their target genes.

title: MADS-box gene expression in lateral primordia, meristems and differentiated tissues of Arabidopsis thaliana roots
abstract: Although MADS-box genes involved in flower and fruit development have been well characterized, the function of MADS-box genes expressed in vegetative structures has yet to be explored. At least seven members of this family are grouped in clades of genes that are preferentially expressed in roots of Arabidopsis thaliana (L.) Heynh.. We report here the cloning of the AGL21 MADS-box gene, which belongs to the ANR1 clade, and the mRNA in situ expression patterns of this and two other root MADS-box genes. AGL17 appears to be a lateral root cap marker in the root tip, and towards the elongation zone this gene is expressed in the epidermal cells. AGL21 is highly expressed in lateral root primordia and it has a punctate expression pattern in the primary root meristem. AGL12 also has a punctate expression pattern in the primary root meristem. AGL12 and AGL21 are also expressed in the central cylinder of differentiated roots and both are expressed in developing embryos. This study, combined with previous phylogenetic analyses, indicates that these MADS-box genes may play distinct regulatory roles during root development.

title: An AGAMOUS-Related MADS-Box Gene, XAL1 (AGL12), Regulates Root Meristem Cell Proliferation and Flowering Transition in Arabidopsis1[W][OA]
abstract: MADS-box genes are key components of the networks that control the transition to flowering and flower development, but their role in vegetative development is poorly understood. This article shows that the sister gene of the AGAMOUS (AG) clade, AGL12, has an important role in root development as well as in flowering transition. We isolated three mutant alleles for AGL12, which is renamed here as XAANTAL1 (XAL1): Two alleles, xal1-1 and xal1-2, are in Columbia ecotype and xal1-3 is in Landsberg erecta ecotype. All alleles have a short-root phenotype with a smaller meristem, lower rate of cell production, and abnormal root apical meristem organization. Interestingly, we also encountered a significantly longer cell cycle in the strongest xal1 alleles with respect to wild-type plants. Expression analyses confirmed the presence of XAL1 transcripts in roots, particularly in the phloem. Moreover, XAL1∷β-glucuronidase expression was specifically up-regulated by auxins in this tissue. In addition, mRNA in situ hybridization showed that XAL1 transcripts were also found in leaves and floral meristems of wild-type plants. This expression correlates with the late-flowering phenotypes of the xal1 mutants grown under long days. Transcript expression analysis suggests that XAL1 is an upstream regulator of SOC, FLOWERING LOCUS T, and LFY. We propose that XAL1 may have similar roles in both root and aerial meristems that could explain the xal1 late-flowering phenotype.

title: An Atlas of Type I MADS Box Gene Expression during Female Gametophyte and Seed Development in Arabidopsis[W]
abstract: Members of the plant type I MADS domain subfamily have been reported to be involved in reproductive development in Arabidopsis (Arabidopsis thaliana). However, from the 61 type I genes in the Arabidopsis genome, only PHERES1, AGAMOUS-LIKE80 (AGL80), DIANA, AGL62, and AGL23 have been functionally characterized, which revealed important roles for these genes during female gametophyte and early seed development. The functions of the other genes are still unknown, despite the fact that the available single T-DNA insertion mutants have been largely investigated. The lack of mutant phenotypes is likely due to a considerable number of recent intrachromosomal duplications in the type I subfamily, resulting in nonfunctional genes in addition to a high level of redundancy. To enable a breakthrough in type I MADS box gene characterization, a framework needs to be established that allows the prediction of the functionality and redundancy of the type I genes. Here, we present a complete atlas of their expression patterns during female gametophyte and seed development in Arabidopsis, deduced from reporter lines containing translational fusions of the genes to green fluorescent protein and β-glucuronidase. All the expressed genes were revealed to be active in the female gametophyte or developing seed, indicating that the entire type I subfamily is involved in reproductive development in Arabidopsis. Interestingly, expression was predominantly observed in the central cell, antipodal cells, and chalazal endosperm. The combination of our expression results with phylogenetic and protein interaction data allows a better identification of putative redundantly acting genes and provides a useful tool for the functional characterization of the type I MADS box genes in Arabidopsis.

title: Phytochemicals from cruciferous plants protect against cancer by modulating carcinogen metabolism.
abstract: Several epidemiologic studies suggest that consumption of cruciferous vegetables may be particularly effective (compared with total fruit and vegetable consumption) in reducing cancer risk at several organ sites. Crucifers that are widely consumed are especially rich in glucosinolates, which are converted by plant myrosinase and gastrointestinal microflora to isothiocyanates. A number of isothiocyanates and a limited number of glucosinolates that were examined effectively block chemical carcinogenesis in animal models. Many isothiocyanates are also potent inducers of phase 2 proteins. Substantial evidence supports the view that phase 2 enzyme induction is a highly effective strategy for reducing susceptibility to carcinogens. This conclusion has recently received strong molecular support from experiments on mice in which the specific transcription factor, nrf2, which is essential for induction of phase 2 proteins, was deleted. In these knock-out mice, the basal levels of phase 2 enzymes are very low and not inducible. Accordingly, these mice are much more susceptible than their wild-type counterparts to benzo[a]pyrene forestomach carcinogenesis and are not protected by phase 2 inducers. These experiments provide very strong evidence for a major role of phase 2 enzymes in controlling the risk of exposure to carcinogens. An increasing number of phase 2 proteins that exert a variety of protective mechanisms are being identified. Thus, in addition to detoxifying electrophiles, these proteins exercise versatile, long-lasting and catalytic antioxidant protection.

title: The Korea Brassica Genome Project: a Glimpse of the Brassica Genome Based on Comparative Genome Analysis With Arabidopsis
abstract: A complete genome sequence provides unlimited information in the sequenced organism as well as in related taxa. According to the guidance of the Multinational Brassica Genome Project (MBGP), the Korea Brassica Genome Project (KBGP) is sequencing chromosome 1 (cytogenetically oriented chromosome #1) of Brassica rapa. We have selected 48 seed BACs on chromosome 1 using EST genetic markers and FISH analyses. Among them, 30 BAC clones have been sequenced and 18 are on the way. Comparative genome analyses of the EST sequences and sequenced BAC clones from Brassica chromosome 1 revealed their homeologous partner regions on the Arabidopsis genome and a syntenic comparative map between Brassica chromosome 1 and Arabidopsis chromosomes. In silico chromosome walking and clone validation have been successfully applied to extending sequence contigs based on the comparative map and BAC end sequences. In addition, we have defined the (peri)centromeric heterochromatin blocks with centromeric tandem repeats, rDNA and centromeric retrotransposons. In-depth sequence analyses of five homeologous BAC clones and an Arabidopsis chromosomal region reveal overall co-linearity, with 82% sequence similarity. The data indicate that the Brassica genome has undergone triplication and subsequent gene losses after the divergence of Arabidopsis and Brassica. Based on in-depth comparative genome analyses, we propose a comparative genomics approach for conquering the Brassica genome. In 2005 we intend to construct an integrated physical map, including sequence information from 500 BAC clones and integration of fingerprinting data and end sequence data of more than 100 000 BAC clones. The sequences have been submitted to GenBank with accession numbers: 10 204 BAC ends of the KBrH library (CW978640–CW988843); KBrH138P04, AC155338; KBrH117N09, AC155337; KBrH097M21, AC155348; KBrH093K03, AC155347; KBrH081N08, AC155346; KBrH080L24, AC155345; KBrH077A05, AC155343; KBrH020D15, AC155340; KBrH015H17, AC155339; KBrH001H24, AC155335; KBrH080A08, AC155344; KBrH004D11, AC155341; KBrH117M18, AC146875; KBrH052O08, AC155342.

title: Genome-wide analysis of the bHLH transcription factor family in Chinese cabbage (Brassica rapa ssp. pekinensis)
abstract: Basic helix-loop-helix (bHLH) transcription factors are widely distributed in eukaryotic organisms and are thought to be one of the largest families of regulatory proteins. This important family of transcriptional regulators plays crucial roles in plant development. However, a systematic analysis of the bHLH transcription factor family has not been reported in Chinese cabbage. In this study, 230 bHLH transcription factors were identified from the whole Chinese cabbage genome and compared with proteins from other representative plants, fungi and metazoans. The Chinese cabbage bHLH (BrabHLH) gene family could be classified into 24 subfamilies. Phylogenetic analysis of BrabHLHs along with bHLHs from Arabidopsis and rice indicated 26 subfamilies. The identification, classification, phylogenetic reconstruction, conserved motifs, chromosome distribution, functional annotation, expression patterns and interaction networks of BrabHLHs were analyzed. Distribution mapping showed that BrabHLHs were non-randomly located on the ten Chinese cabbage chromosomes. One hundred and twenty-four orthologous bHLH genes were identified between Chinese cabbage and Arabidopsis, and the interaction networks of the orthologous genes were constructed in Chinese cabbage. Quantitative RT-PCR analysis showed that expressions of BrabHLH genes varied widely under different abiotic stress treatments for different times. Thus, this comprehensive analysis of BrabHLHs represents a rich resource, aiding the elucidation of the roles of bHLH family members in plant growth and development. Furthermore, the comparative genomics analysis deepened our understanding of the evolution of this gene family after a polyploidy event.

title: Antiquity and evolution of the MADS-box gene family controlling flower development in plants.
abstract: MADS-box genes in plants control various aspects of development and reproductive processes including flower formation. To obtain some insight into the roles of these genes in morphological evolution, we investigated the origin and diversification of floral MADS-box genes by conducting molecular evolutionary genetics analyses. Our results suggest that the most recent common ancestor of today's floral MADS-box genes evolved roughly 650 MYA, much earlier than the Cambrian explosion. They also suggest that the functional classes T (SVP), B (and Bs), C, F (AGL20 or TM3), A, and G (AGL6) of floral MADS-box genes diverged sequentially in this order from the class E gene lineage. The divergence between the class G and E genes apparently occurred around the time of the angiosperm/gymnosperm split. Furthermore, the ancestors of three classes of genes (class T genes, class B/Bs genes, and the common ancestor of the other classes of genes) might have existed at the time of the Cambrian explosion. We also conducted a phylogenetic analysis of MADS-domain sequences from various species of plants and animals and presented a hypothetical scenario of the evolution of MADS-box genes in plants and animals, taking into account paleontological information. Our study supports the idea that there are two main evolutionary lineages (type I and type II) of MADS-box genes in plants and animals.

title: Interaction network of proteins associated with abiotic stress response and development in wheat
abstract: Wheat is the most widely adapted crop to abiotic stresses and considered an excellent system to study stress tolerance in spite of its genetic complexity. Recent studies indicated that several hundred genes are either up- or down-regulated in response to stress treatment. To elucidate the function of some of these genes, an interactome of proteins associated with abiotic stress response and development in wheat was generated using the yeast two-hybrid GAL4 system and specific protein interaction assays. The interactome is comprised of 73 proteins, generating 97 interactions pairs. Twenty-one interactions were confirmed by bimolecular fluorescent complementation in Nicotiana benthamiana. A confidence-scoring system was elaborated to evaluate the significance of the interactions. The main feature of this interactome is that almost all bait proteins along with their interactors were interconnected, creating a spider web-like structure. The interactome revealed also the presence of a “cluster of proteins involved in flowering control” in three- and four-protein interaction loops.This network provides a novel insight into the complex relationships among transcription factors known to play central roles in vernalization, flower initiation and abscisic acid signaling, as well as associations that tie abiotic stress with other regulatory and signaling proteins. This analysis provides useful information in elucidating the molecular mechanism associated with abiotic stress response in plants.

title: MADS-box gene family in rice: genome-wide identification, organization and expression profiling during reproductive development and stress
abstract: BackgroundMADS-box transcription factors, besides being involved in floral organ specification, have also been implicated in several aspects of plant growth and development. In recent years, there have been reports on genomic localization, protein motif structure, phylogenetic relationships, gene structure and expression of the entire MADS-box family in the model plant system, Arabidopsis. Though there have been some studies in rice as well, an analysis of the complete MADS-box family along with a comprehensive expression profiling was still awaited after the completion of rice genome sequencing. Furthermore, owing to the role of MADS-box family in flower development, an analysis involving structure, expression and functional aspects of MADS-box genes in rice and Arabidopsis was required to understand the role of this gene family in reproductive development. genome-wide molecular characterization and microarray-based expression profiling of the genes encoding MADS-box transcription factor family in rice is presented. Using a thorough annotation exercise, 75 MADS-box genes have been identified in rice and categorized into MIKCc, MIKC*, Mα, Mβ and Mγ groups based on phylogeny. Chromosomal localization of these genes reveals that 16 MADS-box genes, mostly MIKCc-type, are located within the duplicated segments of the rice genome, whereas most of the M-type genes, 20 in all, seem to have resulted from tandem duplications. Nine members belonging to the Mβ group, which was considered absent in monocots, have also been identified. The expression profiles of all the MADS-box genes have been analyzed under 11 temporal stages of panicle and seed development, three abiotic stress conditions, along with three stages of vegetative development. Transcripts for 31 genes accumulate preferentially in the reproductive phase, of which, 12 genes are specifically expressed in seeds, and six genes show expression specific to panicle development. Differential expression of seven genes under stress conditions is also evident. An attempt has been made to gain insight into plausible functions of rice MADS-box genes by collating the expression data of functionally validated genes in rice and Arabidopsis.ConclusionOnly a limited number of MADS genes have been functionally validated in rice. A comprehensive annotation and transcriptome profiling undertaken in this investigation adds to our understanding of the involvement of MADS-box family genes during reproductive development and stress in rice and also provides the basis for selection of candidate genes for functional validation studies.

title: BRAD, the genetics and genomics database for Brassica plants
abstract: BackgroundBrassica species include both vegetable and oilseed crops, which are very important to the daily life of common human beings. Meanwhile, the Brassica species represent an excellent system for studying numerous aspects of plant biology, specifically for the analysis of genome evolution following polyploidy, so it is also very important for scientific research. Now, the genome of Brassica rapa has already been assembled, it is the time to do deep mining of the genome data.DescriptionBRAD, the Brassica database, is a web-based resource focusing on genome scale genetic and genomic data for important Brassica crops. BRAD was built based on the first whole genome sequence and on further data analysis of the Brassica A genome species, Brassica rapa (Chiifu-401-42). It provides datasets, such as the complete genome sequence of B. rapa, which was de novo assembled from Illumina GA II short reads and from BAC clone sequences, predicted genes and associated annotations, non coding RNAs, transposable elements (TE), B. rapa genes' orthologous to those in A. thaliana, as well as genetic markers and linkage maps. BRAD offers useful searching and data mining tools, including search across annotation datasets, search for syntenic or non-syntenic orthologs, and to search the flanking regions of a certain target, as well as the tools of BLAST and Gbrowse. BRAD allows users to enter almost any kind of information, such as a B. rapa or A. thaliana gene ID, physical position or genetic marker.ConclusionBRAD, a new database which focuses on the genetics and genomics of the Brassica plants has been developed, it aims at helping scientists and breeders to fully and efficiently use the information of genome data of Brassica plants. BRAD will be continuously updated and can be accessed through http://brassicadb.org.

title: Genome-wide survey and expression analysis of the MADS-box gene family in soybean
abstract: MADS-box genes encode important transcription factors in plants that are involved in many processes during plant growth and development. An investigation of the soybean genome revealed 106 putative MADS-box genes. These genes were classified into two classes, type I and type II, based on phylogenetic analysis. The soybean type II group has 72 members, which is higher than that of Arabidopsis, indicating that soybean type II genes have undergone a higher rate of duplication and/or a lower rate of gene loss after duplication. Soybean MADS-box genes are present on all chromosomes. Like Arabidopsis and rice MADS-box genes, soybean MADS-box genes expanded through tandem gene duplication and segmental duplication events. There are many duplicate genes distributed across the soybean genome, with two genomic regions, i.e., MADS-box gene hotspots, where MADS-box genes with high degrees of similarity are clustered. Analysis of high-throughput sequencing data from soybean at different developmental stages and in different tissues revealed that MADS-box genes are expressed in embryos of various stages and in floral buds. This expression pattern suggests that soybean MADS-box genes play an important role in soybean growth and floral development.

title: Whole-genome survey and characterization of MADS-box gene family in maize and sorghum
abstract: MADS-box genes comprise a large gene family, which codes for transcription factors, and play important functions in various aspects of flowering plant growth and development. However, little is known about the MADS-box genes in maize (Zea mays) and sorghum (Sorghum bicolor). Here, we performed a comprehensive bioinformatics analysis of the MADS-box gene family in the maize and sorghum genomes and identified 75 maize and 65 sorghum MADS-box genes. We subsequently carried out a comparative analysis of these genes, including the gene structure, phylogenetic relationship, conserved protein motifs, gene duplications, chromosomal locations and expression pattern between the two plants. According to these analyses, the MADS-box genes in both maize and sorghum were categorized into five (MIKCC, MIKC*, Mα, Mβ and Mγ) groups, and the MIKCC groups were further divided into 11 subfamilies. In addition, gene duplications of MADS-box genes were also investigated in the maize, sorghum, rice and Arabidopsis genomes. We found a higher percentage of MADS-box gene duplications in the maize and sorghum genomes, which contributed to the expansion of the MADS-box gene family. Furthermore, both tandem and segmental duplications played a major role in the MADS-box gene expansion in maize and sorghum. A survey of maize and sorghum EST sequences indicated that MADS-box genes exhibit a various expression pattern, suggesting diverse and novel functions of MADS-box gene families in the two plants. These results provided a useful reference for selection of candidate MADS-box genes for cloning and further functional analysis in both maize and sorghum.

title: MIKC* MADS-Protein Complexes Bind Motifs Enriched in the Proximal Region of Late Pollen-Specific Arabidopsis Promoters[W]
abstract: The genome of Arabidopsis (Arabidopsis thaliana) encodes over 100 MADS-domain transcription factors, categorized into five phylogenetic subgroups. Most research efforts have focused on just one of these subgroups (MIKCc), whereas the other four remain largely unexplored. Here, we report on five members of the so-called Mδ or Arabidopsis MIKC* (AtMIKC*) subgroup, which are predominantly expressed during the late stages of pollen development. Very few MADS-box genes function in mature pollen, and from this perspective, the AtMIKC* genes are therefore highly exceptional. We found that the AtMIKC* proteins are able to form multiple heterodimeric complexes in planta, and that these protein complexes exhibit a for the MADS-family unusual and high DNA binding specificity in vitro. Compared to their occurrence in promoters genome wide, AtMIKC* binding sites are strongly overrepresented in the proximal region of late pollen-specific promoters. By combining our experimental data with in silico genomics and pollen transcriptomics approaches, we identified a considerable number of putative direct target genes of the AtMIKC* transcription factor complexes in pollen, many of which have known or proposed functions in pollen tube growth. The expression of several of these predicted targets is altered in mutant pollen in which all AtMIKC* complexes are affected, and in vitro germination of this mutant pollen is severely impaired. Our data therefore suggest that the AtMIKC* protein complexes play an essential role in transcriptional regulation during late pollen development.

title: Delayed flowering time in Arabidopsis and Brassica rapa by the overexpression of FLOWERING LOCUS C (FLC) homologs isolated from Chinese cabbage (Brassica rapaL. ssp. pekinensis)
abstract: Chinese cabbage plants remain in the vegetative growth phase until they have experienced prolonged exposure to cold temperature, known as vernalization. This inhibition of flowering is caused by the high levels of FLOWERING LOCUS C (FLC) expression. To increase the product value of Chinese cabbage by inhibiting the floral transition, three genes (BrFLC1, BrFLC2, and BrFLC3) homologous to the AtFLC gene, which encodes a floral repressor, were isolated from the Chinese cabbage ‘Chiifu’. These genes showed high similarity to AtFLC, although the putative BrFLC1 protein contained ten more residues than AtFLC. The BrFLC genes were expressed ubiquitously, except that BrFLC3 was not expressed in roots. BrFLC1 and BrFLC2 showed stronger expression than BrFLC3 in unvernalized and vernalized Chinese cabbage. The expression levels of the three BrFLC genes were lower in an early-flowering Chinese cabbage, suggesting that the BrFLC transcript level was associated with flowering time. Constitutive expression of the BrFLC genes in Arabidopsis significantly delayed flowering, which was also observed in transgenic Chinese cabbage overexpressing BrFLC3. These results suggest that the BrFLC genes act similarly to AtFLC. Our results provide a technique for controlling flowering time in Chinese cabbage and other crops to produce high yields of vegetative tissues.

title: Gene duplication and evolutionary novelty in plants.
abstract: Duplication is a prominent feature of plant genomic architecture. This has led many researchers to speculate that gene duplication may have played an important role in the evolution of phenotypic novelty within plants. Until recently, however, it was difficult to make this connection. We are now beginning to understand how duplication has contributed to adaptive evolution in plants. In this review we introduce the sources of gene duplication and predictions of the various fates of duplicates. We also highlight several recent and pertinent examples from the literature. These examples demonstrate the importance of the functional characteristics of genes and the source of duplication in influencing evolutionary outcome.

title: The collapse of gene complement following whole genome duplication
abstract: BackgroundGenome amplification through duplication or proliferation of transposable elements has its counterpart in genome reduction, by elimination of DNA or by gene inactivation. Whether loss is primarily due to excision of random length DNA fragments or the inactivation of one gene at a time is controversial. Reduction after whole genome duplication (WGD) represents an inexorable collapse in gene complement.ResultsWe compare fifteen genomes descending from six eukaryotic WGD events 20-450 Mya. We characterize the collapse over time through the distribution of runs of reduced paralog pairs in duplicated segments. Descendant genomes of the same WGD event behave as replicates. Choice of paralog pairs to be reduced is random except for some resistant regions of contiguous pairs. For those paralog pairs that are reduced, conserved copies tend to concentrate on one chromosome.ConclusionsBoth the contiguous regions of reduction-resistant pairs and the concentration of runs of single copy genes on a single chromosome are evidence of transcriptional co-regulation, dosage sensitivity or other functional interaction constraining the reduction process. These constraints and their evolution over time show a consistent pattern across evolutionary domains and a highly reproducible pattern, as replicates, for the several descendants of a single WGD.

title: Biased Gene Fractionation and Dominant Gene Expression among the Subgenomes of Brassica rapa
abstract: Polyploidization, both ancient and recent, is frequent among plants. A “two-step theory" was proposed to explain the meso-triplication of the Brassica “A" genome: Brassica rapa. By accurately partitioning of this genome, we observed that genes in the less fractioned subgenome (LF) were dominantly expressed over the genes in more fractioned subgenomes (MFs: MF1 and MF2), while the genes in MF1 were slightly dominantly expressed over the genes in MF2. The results indicated that the dominantly expressed genes tended to be resistant against gene fractionation. By re-sequencing two B. rapa accessions: a vegetable turnip (VT117) and a Rapid Cycling line (L144), we found that genes in LF had less non-synonymous or frameshift mutations than genes in MFs; however mutation rates were not significantly different between MF1 and MF2. The differences in gene expression patterns and on-going gene death among the three subgenomes suggest that “two-step" genome triplication and differential subgenome methylation played important roles in the genome evolution of B. rapa.

title: Altered Patterns of Fractionation and Exon Deletions in Brassica rapa Support a Two-Step Model of Paleohexaploidy
abstract: The genome sequence of the paleohexaploid Brassica rapa shows that fractionation is biased among the three subgenomes and that the least fractionated subgenome has approximately twice as many orthologs as its close (and relatively unduplicated) relative Arabidopsis than had either of the other two subgenomes. One evolutionary scenario is that the two subgenomes with heavy gene losses (I and II) were in the same nucleus for a longer period of time than the third subgenome (III) with the fewest gene losses. This “two-step” hypothesis is essentially the same as that proposed previously for the eudicot paleohexaploidy; however, the more recent nature of the B. rapa paleohexaploidy makes this model more testable. We found that subgenome II suffered recent small deletions within exons more frequently than subgenome I, as would be expected if the genes in subgenome I had already been near maximally fractionated before subgenome III was introduced. We observed that some sequences, before these deletions, were flanked by short direct repeats, a unique signature of intrachromosomal illegitimate recombination. We also found, through simulations, that short—single or two-gene—deletions appear to dominate the fractionation patterns in B. rapa. We conclude that the observed patterns of the triplicated regions in the Brassica genome are best explained by a two-step fractionation model. The triplication and subsequent mode of fractionation could influence the potential to generate morphological diversity—a hallmark of the Brassica genus.

title: Genome-wide comparative analysis of the Brassica rapa gene space reveals genome shrinkage and differential loss of duplicated genes after whole genome triplication
abstract: BackgroundBrassica rapa is one of the most economically important vegetable crops worldwide. Owing to its agronomic importance and phylogenetic position, B. rapa provides a crucial reference to understand polyploidy-related crop genome evolution. The high degree of sequence identity and remarkably conserved genome structure between Arabidopsis and Brassica genomes enables comparative tiling sequencing using Arabidopsis sequences as references to select the counterpart regions in B. rapa, which is a strong challenge of structural and comparative crop genomics.ResultsWe assembled 65.8 megabase-pairs of non-redundant euchromatic sequence of B. rapa and compared this sequence to the Arabidopsis genome to investigate chromosomal relationships, macrosynteny blocks, and microsynteny within blocks. The triplicated B. rapa genome contains only approximately twice the number of genes as in Arabidopsis because of genome shrinkage. Genome comparisons suggest that B. rapa has a distinct organization of ancestral genome blocks as a result of recent whole genome triplication followed by a unique diploidization process. A lack of the most recent whole genome duplication (3R) event in the B. rapa genome, atypical of other Brassica genomes, may account for the emergence of B. rapa from the Brassica progenitor around 8 million years ago.ConclusionsThis work demonstrates the potential of using comparative tiling sequencing for genome analysis of crop species. Based on a comparative analysis of the B. rapa sequences and the Arabidopsis genome, it appears that polyploidy and chromosomal diploidization are ongoing processes that collectively stabilize the B. rapa genome and facilitate its evolution.

title: MIKC-type MADS-domain proteins: structural modularity, protein interactions and network evolution in land plants.
abstract: MIKC-type proteins represent a class of MADS-domain transcription factors and are defined by a unique domain structure: in addition to the highly conserved DNA-binding MADS-domain, they have three other domains ('I', 'K' and 'C'), with the keratin-like K-domain being the most highly conserved and characteristic one. The number and functional diversity of MIKC-type proteins increased considerably during land plant evolution, culminating in higher flowering plants, where they dominate the control of reproductive development from early to late stages. We wonder how one special class of proteins became important in the control of essentially all stages of a morphogenetic process. All MADS-domain proteins appear to bind to DNA as homo- or heterodimers and may function as part of ternary transcription factor complexes involving non-MADS-domain proteins. Only MIKC-type proteins, however, generate complex intrafamily interaction networks. These are based on the special potential of MIKC-type proteins to form complexes involving more than two homologous proteins constituting transcriptional regulators. We speculate that the potential to form heteromultimers of homologous proteins was achieved by the acquisition of the K-domain during evolution. There is emerging evidence that organismal complexity arises from progressively more elaborate regulation of gene expression. We hypothesize that combinatorial multimer formation of MIKC-type MADS-domain proteins facilitated an unusually efficient and rapid functional diversification based on gene duplication, sequence divergence and fixation. This 'networking' may have enabled a more sophisticated transcriptional control of target genes which was recruited for controlling increasingly complex and diverse developmental pathways during the rapid origin and diversification of plant reproductive structures. Therefore, MIKC-type proteins may owe their evolutionary 'success' and present-day developmental importance in part to their modular domain structure. Investigating the evolution of MIKC-type genes may thus help to better understand origin and diversification of gene regulatory networks.

title: Molecular cloning of SVP: a negative regulator of the floral transition in Arabidopsis.
abstract: Flowering time mutants represent genetic functions in control of the floral transition, an important developmental phase switch in the life cycle of higher plants. Many such mutants have been identified and characterized, particular in Arabidopsis. Here we describe the identification and initial characterization of a new early flowering mutant of Arabidopsis. The corresponding gene, SVP (SHORT VEGETATIVE PHASE), was cloned through transposon tagging and represents a new member of the MADS-box gene family of transcription factors. Analysis of its transcriptional activity revealed the presence of differently sized transcripts that were confined to vegetative tissues and floral primordia and absent from developed flowers and siliques. The function of SVP as a repressor of the floral transition is discussed.

title: AGAMOUS-LIKE 24, a dosage-dependent mediator of the flowering signals
abstract: The most dramatic phase change in plants is the transition from vegetative to reproductive growth. This flowering process is regulated by several interacting pathways that monitor both the developmental state of the plants and environmental cues such as light and temperature. The flowering-time genes FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), together with the floral meristem identity gene LEAFY (LFY), are three essential regulators integrating floral signals from multiple pathways in Arabidopsis thaliana. Part of the crosstalk among these genes is mediated by a putative transcription factor, AGAMOUS-LIKE 24 (AGL24). This gene is gradually activated in shoot apical meristems during the floral transition and later located in the whole zone of both inflorescence and floral meristems. Loss and reduction of AGL24 activity by double-stranded RNA-mediated interference result in late flowering, whereas constitutive overexpression of AGL24 causes precocious flowering. The correlation between the level of AGL24 accumulation and the alteration of flowering time suggests that AGL24 is a dosage-dependent flowering promoter. Analysis of AGL24 expression in various flowering-time mutants shows that it is regulated in several floral inductive pathways. Further genetic analyses of epistasis indicate that AGL24 may act downstream of SOC1 and upstream of LFY.

title: Evolution of class B floral homeotic proteins: obligate heterodimerization originated from homodimerization.
abstract: The class B floral homeotic genes from the higher eudicot model systems Arabidopsis and Antirrhinum are involved in specifying the identity of petals and stamens during flower development. These genes exist in two different types termed DEF- and GLO-like genes. The proteins encoded by the class B genes are stable and functional in the cell only as heterodimeric complexes of a DEF- and a GLO-like protein. In line with this, heterodimerization is obligate for DNA binding in vitro. The genes whose products have to heterodimerize to be stable and functional are each other's closest relatives within their genomes. This suggests that the respective genes originated by gene duplication, and that heterodimerization is of relative recent origin and evolved from homodimerization. To test this hypothesis we have investigated the dimerization behavior of putative B proteins from phylogenetic informative taxa, employing electrophoretic mobility shift assays and the yeast two-hybrid system. We find that an ancestral B protein from the gymnosperm Gnetum gnemon binds DNA in a sequence-specific manner as a homodimer. Of the two types of B proteins from the monocot Lilium regale, the GLO-like protein is still able to homodimerize, whereas the DEF-like protein binds to DNA only as a heterodimeric complex with the GLO-like protein. These data suggest that heterodimerization evolved in two steps after a gene duplication that gave rise to DEF- and GLO-like genes. Heterodimerization may have originated after the gymnosperm-angiosperm split about 300 MYA but before the monocot-eudicot split 140-200 MYA. Heterodimerization may have become obligate for both types of flowering plant B proteins in the eudicot lineage after the monocot-eudicot split.

title: A large family of TM3 MADS-box cDNAs in Pinus radiata includes two members with deletions of the conserved K domain
abstract: Angiosperm flower organ formation is regulated by three conserved groups of MADS-box genes, but the function of a fourth conserved group, the TM3 orthologues, remains unclear. We report the isolation of six different MADS-box cDNAs from male cones of the gymnosperm Pinus radiata. All are orthologues of TM3 and two encode proteins with different sized deletions within the conserved K domain. Both the deletions and the large number of paralogous duplications within one clade of MADS-box genes are unusual and indicate that this gene family may have been under selection to evolve new functions in conifers.

title: MADS-box genes reveal that gnetophytes are more closely related to conifers than to flowering plants.
abstract: The evolutionary origin of the angiosperms (flowering plants sensu stricto) is still enigmatic. Answers to the question of angiosperm origins are intimately connected to the identification of their sister group among extinct and extant taxa. Most phylogenetic analyses based on morphological data agree that among the groups of extant seed plants, the gnetophytes are the sister group of the angiosperms. According to this view, angiosperms and gnetophytes are the only extant members of a clade called "anthophytes" to emphasize their shared possession of flower-like reproductive structures. However, most phylogeny reconstructions based on molecular data so far did not support an anthophyte clade, but also could not clarify the case because support for alternative groupings has been weak or controversial. We have isolated 13 different homologs of MADS-type floral homeotic genes from the gnetophyte Gnetum gnemon. Five of these genes fall into monophyletic gene clades also comprising putatively orthologous genes from flowering plants and conifers, among them orthologs of floral homeotic B and C function genes. Within these clades the Gnetum genes always form distinct subclades together with the respective conifer genes, to the exclusion of the angiosperm genes. This provides strong molecular evidence for a sister-group relationship between gnetophytes and conifers, which is in contradiction to widely accepted interpretations of morphological data for almost a century. Our phylogeny reconstructions and the outcome of expression studies suggest that complex features such as flower-like reproductive structures and double-fertilization arose independently in gnetophytes and angiosperms.

title: Regulation and function of SOC1, a flowering pathway integrator.
abstract: SOC1, encoding a MADS box transcription factor, integrates multiple flowering signals derived from photoperiod, temperature, hormone, and age-related signals. SOC1 is regulated by two antagonistic flowering regulators, CONSTANS (CO) and FLOWERING LOCUS C (FLC), which act as floral activator and repressor, respectively. CO activates SOC1 mainly through FT but FLC represses SOC1 by direct binding to the promoter. SOC1 is also activated by an age-dependent mechanism in which SPL9 and microRNA156 are involved. When SOC1 is induced at the shoot apex, SOC1 together with AGL24 directly activates LEAFY (LFY), a floral meristem identity gene. APETALA1 (AP1), activated mainly by FT, is also necessary to establish and maintain flower meristem identity. When LFY and AP1 are established, flower development occurs at the anlagen of shoot apical meristem according to the ABC model. During early flower development, AP1 activates the A function and represses three redundantly functioning flowering time genes, SOC1, AGL24, and SVP to prevent floral reversion. During late flower development, such repression is also necessary to activate SEPALATA3 (SEP3) which is a coactivator of B and C function genes with LFY, otherwise SEP3 is suppressed by SOC1, AGL24, and SVP. Therefore, SOC1 is necessary to prevent premature differentiation of the floral meristem.

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: SEPALLATA3: the 'glue' for MADS box transcription factor complex formation
abstract: BackgroundPlant MADS box proteins play important roles in a plethora of developmental processes. In order to regulate specific sets of target genes, MADS box proteins dimerize and are thought to assemble into multimeric complexes. In this study a large-scale yeast three-hybrid screen is utilized to provide insight into the higher-order complex formation capacity of the Arabidopsis MADS box family. SEPALLATA3 (SEP3) has been shown to mediate complex formation and, therefore, special attention is paid to this factor in this study.ResultsIn total, 106 multimeric complexes were identified; in more than half of these at least one SEP protein was present. Besides the known complexes involved in determining floral organ identity, various complexes consisting of combinations of proteins known to play a role in floral organ identity specification, and flowering time determination were discovered. The capacity to form this latter type of complex suggests that homeotic factors play essential roles in down-regulation of the MADS box genes involved in floral timing in the flower via negative auto-regulatory loops. Furthermore, various novel complexes were identified that may be important for the direct regulation of the floral transition process. A subsequent detailed analysis of the APETALA3, PISTILLATA, and SEP3 proteins in living plant cells suggests the formation of a multimeric complex in vivo.ConclusionsOverall, these results provide strong indications that higher-order complex formation is a general and essential molecular mechanism for plant MADS box protein functioning and attribute a pivotal role to the SEP3 'glue' protein in mediating multimerization.

title: Type I MADS-box genes have experienced faster birth-and-death evolution than type II MADS-box genes in angiosperms
abstract: Plant MADS-box genes form a large gene family for transcription factors and are involved in various aspects of developmental processes, including flower development. They are known to be subject to birth-and-death evolution, but the detailed features of this mode of evolution remain unclear. To have a deeper insight into the evolutionary pattern of this gene family, we enumerated all available functional and nonfunctional (pseudogene) MADS-box genes from the Arabidopsis and rice genomes. Plant MADS-box genes can be classified into types I and II genes on the basis of phylogenetic analysis. Conducting extensive homology search and phylogenetic analysis, we found 64 presumed functional and 37 nonfunctional type I genes and 43 presumed functional and 4 nonfunctional type II genes in Arabidopsis. We also found 24 presumed functional and 6 nonfunctional type I genes and 47 presumed functional and 1 nonfunctional type II genes in rice. Our phylogenetic analysis indicated there were at least about four to eight type I genes and ≈15–20 type II genes in the most recent common ancestor of Arabidopsis and rice. It has also been suggested that type I genes have experienced a higher rate of birth-and-death evolution than type II genes in angiosperms. Furthermore, the higher rate of birth-and-death evolution in type I genes appeared partly due to a higher frequency of segmental gene duplication and weaker purifying selection in type I than in type II genes.

title: The MADS domain factors AGL15 and AGL18 act redundantly as repressors of the floral transition in Arabidopsis.
abstract: The developmental roles of AGL15 and AGL18, members of the AGL15-like clade of MADS domain regulatory factors, have not been defined previously. Analysis of transgenic Arabidopsis plants showed that overexpression of AGL18 produces the same phenotypic changes as overexpression of AGL15, and the two genes have partially overlapping expression patterns. Functional redundancy was confirmed through analysis of loss-of-function mutants. agl15 agl18 double mutants, but not single mutants, flower early under non-inductive conditions, indicating that AGL15 and AGL18 act in a redundant fashion as repressors of the floral transition. Further genetic analyses and expression studies were used to examine the relationship between AGL15 and AGL18 activity and other regulators of the floral transition. AGL15 and AGL18 act upstream of the floral integrator FT, and a combination of agl15 and agl18 mutations partially suppresses defects in the photoperiod pathway. agl15 agl18 mutations show an additive relationship with mutations in genes encoding other MADS domain floral repressors, and further acceleration of flowering is seen in triple and quadruple mutants under both inductive and non-inductive conditions. Thus, flowering time is determined by the additive effect of multiple MADS domain floral repressors, with important contributions from AGL15 and AGL18.

title: AGAMOUS-LIKE 17, a novel flowering promoter, acts in a FT-independent photoperiod pathway.
abstract: The photoperiod pathway is a genetically conserved pathway that affects flowering in distantly related angiosperms. Here we report a novel flowering promoter AGAMOUS-LIKE 17 (AGL17) acting in the photoperiod pathway of Arabidopsis. AGL17 transcripts were detectable in various plant organs with the highest expression in the root. Under long-day conditions, expression of AGL17 gradually increased in the aerial part of seedlings during the floral transition. Overexpression of AGL17 caused early flowering, while loss of function of AGL17 exhibited late flowering, particularly under long days. Analysis of AGL17 expression in various flowering-time mutants showed that its transcripts were significantly reduced in the photoperiod pathway mutant co-1. Correspondingly, AGL17 expression was upregulated in transgenic plants overexpressing CONSTANS (CO) and also when CO activity was induced by light. Genetic analysis further showed that overexpression of AGL17 could partially suppress the late flowering of co-1. These results suggest that AGL17 acts to promote flowering and is positively controlled by the photoperiod pathway regulator CO. In contrast, another target of CO, FLOWERING LOCUS T (FT), did not affect AGL17 expression, and vice versa. The expression of two floral meristem identity genes LEAFY (LFY) and APETALA1 (AP1) decreased in agl17-1, while LFY and AP1 could be rapidly induced by AGL17 using a functional estradiol-inducible system. These findings indicate that AGL17 ultimately promotes flowering via regulation of LFY and AP1.

title: A novel MADS-box gene subfamily with a sister-group relationship to class B floral homeotic genes
abstract: Class B floral homeotic genes specify the identity of petals and stamens during the development of angiosperm flowers. Recently, putative orthologs of these genes have been identified in different gymnosperms. Together, these genes constitute a clade, termed B genes. Here we report that diverse seed plants also contain members of a hitherto unknown sister clade of the B genes, termed Bsister (Bs) genes. We have isolated members of the Bs clade from the gymnosperm Gnetum gnemon, the monocotyledonous angiosperm Zea mays and the eudicots Arabidopsis thaliana and Antirrhinum majus. In addition, MADS-box genes from the basal angiosperm Asarum europaeum and the eudicot Petunia hybrida were identified as Bs genes. Comprehensive expression studies revealed that Bs genes are mainly transcribed in female reproductive organs (ovules and carpel walls). This is in clear contrast to the B genes, which are predominantly expressed in male reproductive organs (and in angiosperm petals). Our data suggest that the Bs genes played an important role during the evolution of the reproductive structures in seed plants. The establishment of distinct B and Bs gene lineages after duplication of an ancestral gene may have accompanied the evolution of male microsporophylls and female megasporophylls 400–300 million years ago. During flower evolution, expression of Bs genes diversified, but the focus of expression remained in female reproductive organs. Our findings imply that a clade of highly conserved close relatives of class B floral homeotic genes has been completely overlooked until recently and awaits further evaluation of its developmental and evolutionary importance. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00438-001-0615-8.

title: Ancestry of plant MADS-box genes revealed by bryophyte (Physcomitrella patens) homologues.
abstract: Three MADS-box cDNA clones and two corresponding genomic sequences (gDNAs) have been isolated from the bryophyte Physcomitrella patens and sequenced. Our findings indicate that the genes may be expressed in a tissue- or age-specific manner, and that expression of one of them is regulated by an alternative splicing mechanism. Conceptual translation of the clones reveals that the encoded MADS-domain proteins have the typical plant-domain pattern (MIKC). Additionally, there is a high degree of conservation of intron number and positions between angiosperm MADS-box genes and the moss loci. These observations confirm the homology of moss and higher plant MADS-box genes. We conclude that the MIKC pattern evolved in MADS-box genes after the separation of the plant lineage from that of fungi and animals, and that it must have been present in the common ancestor of mosses, ferns and seed plants. Therefore it evolved at least 400 million yr ago. Phylogenetic analysis of a large subset of the sequenced plant MADS-box genes, incorporating those from P. patens, indicates that the bryophyte genes are not orthologues of spermatophyte genes belonging to any of the well recognized higher plant gene subfamilies. This conclusion accords well with reports that the known fern MADS-box genes also comprise subfamilies distinct from those of higher plants. Therefore we tentatively propose that the gene duplication and diversification events that created the MADS-box gene subfamilies, discernible in extant angiosperm and other spermatophyte groups, occurred after separation of the moss and fern lineages from the lineage which produced the higher plants.

title: Direct interaction of AGL24 and SOC1 integrates flowering signals in Arabidopsis
abstract: During the transition from vegetative to reproductive growth, the shoot meristem of flowering plants acquires the inflorescence identity to generate flowers rather than vegetative tissues. An important regulator that promotes the inflorescence identity in Arabidopsis is AGAMOUS-LIKE 24 (AGL24), a MADS-box transcription factor. Using a functional estradiol-inducible system in combination with microarray analysis, we identified AGL24-induced genes, including SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), a floral pathway integrator. Chromatin immunoprecipitation (ChIP) analysis of a functional AGL24-6HA-tagged line revealed in vivo binding of AGL24-6HA to the regulatory region of SOC1. Mutagenesis of the AGL24 binding site in the SOC1 promoter decreased ProSOC1:GUS expression and compromised SOC1 function in promoting flowering. Our results show that SOC1 is one of the direct targets of AGL24, and that SOC1 expression is upregulated by AGL24 at the shoot apex at the floral transitional stage. ChIP assay using a functional SOC1-9myc-tagged line and promoter mutagenesis analysis also revealed in vivo binding of SOC1-9myc to the regulatory regions of AGL24 and upregulation of AGL24 at the shoot apex by SOC1. Furthermore, we found that as in other flowering genetic pathways, the effect of gibberellins on flowering under short-day conditions was mediated by the interaction between AGL24 and SOC1. These observations suggest that during floral transition, a positive-feedback loop conferred by direct transcriptional regulation between AGL24 and SOC1 at the shoot apex integrates flowering signals.

title: FLOWERING LOCUS C (FLC) regulates development pathways throughout the life cycle of Arabidopsis
abstract: FLOWERING LOCUS C (FLC) has a key role in the timing of the initiation of flowering in Arabidopsis. FLC binds and represses two genes that promote flowering, FT and SOC1. We show that FLC binds to many other genes, indicating that it has regulatory roles other than the repression of flowering. We identified 505 FLC binding sites, mostly located in the promoter regions of genes and containing at least one CArG box, the motif known to be associated with MADS-box proteins such as FLC. We examined 40 of the target genes, and 20 showed increased transcript levels in an flc mutant compared with the wild type. Five genes showed decreased expression in the mutant, indicating that FLC binding can result in either transcriptional repression or activation. The genes we identified as FLC targets are involved in developmental pathways throughout the life history of the plant, many of which are associated with reproductive development. FLC is also involved in vegetative development, as evidenced by its binding to SPL15, delaying the progression from juvenile to adult phase. Some of the FLC target genes are also bound by two other MADS-box proteins, AP1 and SEP3, suggesting that MADS-box genes may operate in a network of control at different stages of the life cycle, many ultimately contributing to the development of the reproductive phase of the plant.

title: JOINTLESS is a MADS-box gene controlling tomato flower abscissionzone development
abstract: Abscission is a universal and dynamic process in plants whereby organssuch as leaves, flowers and fruit are shed, both during normal development,and in response to tissue damage and stress. Shedding occursby separation of cells in anatomically distinct regions of the plant, calledabscission zones (AZs). During abscission, the plant hormone ethylene stimulatescells to produce enzymes that degrade the middle lamella between cells inthe AZ. The physiology and regulation of abscission at fully developed AZsis well known, but the molecular biology underlying theirdevelopment is not. Here we report the first isolation of a gene directlyinvolved in the development of a functional plant AZ. Tomato plants with the jointless mutation fail to develop AZs on their pedicelsand so abscission of flowers or fruit does not occur normally. We identify JOINTLESS as a new MADS-box gene in a distinct phylogenetic clade separatefrom those functioning in floral organs. We propose that a deletion in JOINTLESS accounts for the failure of activation of pedicel AZ developmentin jointless tomato plants.

title: Isolation and molecular characterization of a new vegetative MADS-box gene from Solanum tuberosum L.
abstract: A cDNA clone, STMADS11, encoding a new MADS-box protein was isolated from Solanum tuberosum L. (potato). Expression of STMADS11 was found in all vegetative organs of the plant, but not in floral tissues. The expression was also detected in all developmental stages, from tuber sprouts to mature plants, reaching a maximum in well-developed organs. However, the level of STMADS11 mRNA was low in tissues such as resting tuber or sprouts developed in the cold, where the metabolic activity is reduced. “In situ” hybridizations performed on leaf and stem sections showed that the STMADS11 transcript is mainly associated with vascular bundles. Cladistic analysis arising from amino acid sequence comparison revealed that STMADS11 shows the highest similarity to STMADS16, another vegetative MADS-box gene from potato, and to the previously reported “orphan” genes AGL15 and AGL17 from Arabidopsis thaliana. Possible implications of these data in relation to STMADS11 function are discussed.

title: Nucleotide sequences of novel potato (Solanum tuberosum L.) MADS-box cDNAs and their expression in vegetative organs.
abstract: Two similar, apparently full-length potato cDNAs, POTM1-1 and POTM1-2, from an early tuber cDNA library have been identified and sequenced. These two cDNAs encode 250 identical amino acids (aa) including 56 aa of the MADS-box domain and 53 aa of the K-box domain, indicating that they may function as transcription factors. The transcripts of these cDNAs accumulate abundantly in vegetative organs, suggesting that these novel MADS genes may be involved in vegetative organ development of potato.

title: Tomato flower abnormalities induced by low temperatures are associated with changes of expression of MADS-Box genes
abstract: Flower and fruit development in tomato (Lycopersicon esculentum Mill. ) were severely affected when plants were grown at low temperatures, displaying homeotic and meristic transformations and alterations in the fusion pattern of the organs. Most of these homeotic transformations modified the identity of stamens and carpels, giving rise to intermediate organs. Complete homeotic transformations were rarely found and always affected organs of the reproductive whorls. Meristic transformations were also commonly observed in the reproductive whorls, which developed with an excessive number of organs. Scanning electron microscopy revealed that meristic transformations take place very early in the development of the flower and are related to a significant increase in the floral meristem size. However, homeotic transformations should occur later during the development of the organ primordia. Steady-state levels of transcripts corresponding to tomato MADS-box genes TM4, TM5, TM6, and TAG1 were greatly increased by low temperatures and could be related to these flower abnormalities. Moreover, in situ hybridization analyses showed that low temperatures also altered the stage-specific expression of TM4.

title: FLC or not FLC: the other side of vernalization.
abstract: Vernalization is the promotion of the competence for flowering by long periods of low temperatures such as those typically experienced during winters. In Arabidopsis, the vernalization response is, to a large extent, mediated by the repression of the floral repressor FLC, and the stable epigenetic silencing of FLC after cold treatments is essential for vernalization. In addition to FLC, other vernalization targets exist in Arabidopsis. In grasses, vernalization seems to be entirely independent of FLC. Here, the current understanding of FLC-independent branches of the vernalization pathway in Arabidopsis and vernalization without FLC in grasses is discussed. This review focuses on the role of AGL19, AGL24, and the MAF genes in Arabidopsis. Interestingly, vernalization acts through related molecular machineries on distinct targets. In particular, protein complexes similar to Drosophila Polycomb Repressive Complex 2 play a prominent role in establishing an epigenetic cellular memory for cold-regulated expression states of AGL19 and FLC. Finally, the similar network topology of the apparently independently evolved vernalization pathways of grasses and Arabidopsis is discussed.

title: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.
abstract: The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved for the alignment of divergent protein sequences. Firstly, individual weights are assigned to each sequence in a partial alignment in order to down-weight near-duplicate sequences and up-weight the most divergent ones. Secondly, amino acid substitution matrices are varied at different alignment stages according to the divergence of the sequences to be aligned. Thirdly, residue-specific gap penalties and locally reduced gap penalties in hydrophilic regions encourage new gaps in potential loop regions rather than regular secondary structure. Fourthly, positions in early alignments where gaps have been opened receive locally reduced gap penalties to encourage the opening up of new gaps at these positions. These modifications are incorporated into a new program, CLUSTAL W which is freely available.

title: [GSDS: a gene structure display server].
abstract: We developed a web server GSDS (Gene Structure Display Server) for drawing gene structure schematic diagrams. Users can submit three types of dataCDS and genomic sequences, NCBI GenBank accession numbers or GIs, exon positions on a gene. GSDS uses this information to obtain the gene structure and draw diagram for it. Users can also designate some special regions to mark on the gene structure diagram. The output result will be PNG or SVG format picture. The corresponding sequence will be shown in a new window by clicking the picture in PNG format. A Chinese version for the main page is also built. The GSDS is available on

title: The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.
abstract: CLUSTAL X is a new windows interface for the widely-used progressive multiple sequence alignment program CLUSTAL W. The new system is easy to use, providing an integrated system for performing multiple sequence and profile alignments and analysing the results. CLUSTAL X displays the sequence alignment in a window on the screen. A versatile sequence colouring scheme allows the user to highlight conserved features in the alignment. Pull-down menus provide all the options required for traditional multiple sequence and profile alignment. New features include: the ability to cut-and-paste sequences to change the order of the alignment, selection of a subset of the sequences to be realigned, and selection of a sub-range of the alignment to be realigned and inserted back into the original alignment. Alignment quality analysis can be performed and low-scoring segments or exceptional residues can be highlighted. Quality analysis and realignment of selected residue ranges provide the user with a powerful tool to improve and refine difficult alignments and to trap errors in input sequences. CLUSTAL X has been compiled on SUN Solaris, IRIX5.3 on Silicon Graphics, Digital UNIX on DECstations, Microsoft Windows (32 bit) for PCs, Linux ELF for x86 PCs, and Macintosh PowerMac.

title: MEME: discovering and analyzing DNA and protein sequence motifs
abstract: MEME (Multiple EM for Motif Elicitation) is one of the most widely used tools for searching for novel ‘signals’ in sets of biological sequences. Applications include the discovery of new transcription factor binding sites and protein domains. MEME works by searching for repeated, ungapped sequence patterns that occur in the DNA or protein sequences provided by the user. Users can perform MEME searches via the web server hosted by the National Biomedical Computation Resource () and several mirror sites. Through the same web server, users can also access the Motif Alignment and Search Tool to search sequence databases for matches to motifs encoded in several popular formats. By clicking on buttons in the MEME output, users can compare the motifs discovered in their input sequences with databases of known motifs, search sequence databases for matches to the motifs and display the motifs in various formats. This article describes the freely accessible web server and its architecture, and discusses ways to use MEME effectively to find new sequence patterns in biological sequences and analyze their significance.

title: Genome-wide identification and expression analysis of calcium-dependent protein kinase in maize
abstract: BackgroundCalcium-dependent protein kinases (CDPKs) have been shown to play important roles in various physiological processes, including plant growth and development, abiotic and biotic stress responses and plant hormone signaling in plants.ResultsIn this study, we performed a bioinformatics analysis of the entire maize genome and identified 40 CDPK genes. Phylogenetic analysis indicated that 40 ZmCPKs can be divided into four groups. Most maize CDPK genes exhibited different expression levels in different tissues and developmental stages. Twelve CDPK genes were selected to respond to various stimuli, including salt, drought and cold, as well as ABA and H2O2. Expression analyses suggested that maize CDPK genes are important components of maize development and multiple transduction pathways.ConclusionHere, we present a genome-wide analysis of the CDPK gene family in maize for the first time, and this genomic analysis of maize CDPK genes provides the first step towards a functional study of this gene family in maize.

title: Genome-wide expression profiles of contrasting inbred lines of Chinese cabbage, Chiifu and Kenshin, under temperature stress
abstract: Abiotic stresses such as extreme temperatures frequently limit plant growth and productivity of major crop species. To identify temperature-stress responsive genes in Brassica rapa ssp. pekinensis, we conducted a comparative transcriptome analysis of two contrasting genotypes, the inbred lines Chiifu and Kenshin, under and heat-shock conditions. Of 23,929 unigenes deposited on a Br24K microarray, 17,603 genes were significantly expressed. Chiifu expression profiles were more prevalent under , and those of Kenshin were more prevalent under heat-shock. Previously characterized genes, with little exception, show similar changes to temperature stress. We found that a relatively large number of genes were expressed in response to temperature stress and genotype, amounting to 24 and 126 genes from and heat-shock conditions, respectively. These genes could be used in molecular marker development for either high or low temperature-resistant Brassica spp. Only ten genes, including TCH4 and CBL1, were up-regulated in both genotypes under all stress treatments, implying possible candidates for broad spectrum, stress-resistant crop production.

title: A comparison of normalization methods for high density oligonucleotide array data based on variance and bias
abstract: When running experiments that involve multiple high density oligonucleotide arrays, it is important to remove sources of variation between arrays of non-biological origin. Normalization is a process for reducing this variation. It is common to see non-linear relations between arrays and the standard normalization provided by Affymetrix does not perform well in these situations.



We present three methods of performing normalization at the probe intensity level. These methods are called complete data methods because they make use of data from all arrays in an experiment to form the normalizing relation. These algorithms are compared to two methods that make use of a baseline array: a one number scaling based algorithm and a method that uses a non-linear normalizing relation by comparing the variability and bias of an expression measure. Two publicly available datasets are used to carry out the comparisons. The simplest and quickest complete data method is found to perform favorably.


AVAILABILITY
Software implementing all three of the complete data normalization methods is available as part of the R package Affy, which is a part of the Bioconductor project http://www.bioconductor.org.



Additional figures may be found at http://www.stat.berkeley.edu/~bolstad/normalize/index.html