Lucia Colombo

Molecular and Phylogenetic Analyses of the Complete MADS-Box Transcription Factor Family in Arabidopsis: New Openings to the MADS World

MADS-box transcription factors are key regulators of several plant development processes. Analysis of the complete Arabidopsis genome sequence revealed 107 genes encoding MADS-box proteins, of which 84% are of unknown function. Here, we provide a complete overview of this family, describing the gene structure, gene expression, genome localization, protein motif organization, and phylogenetic relationship of each member. We have divided this transcription factor family into five groups (named MIKC, Mα, Mβ, Mγ, and Mδ) based on the phylogenetic relationships of the conserved MADS-box domain. This study provides a solid base for functional genomics studies into this important family of plant regulatory genes, including the poorly characterized group of M-type MADS-box proteins. MADS-box genes also constitute an excellent system with which to study the evolution of complex gene families in higher plants.

Comprehensive Interaction Map of the Arabidopsis MADS Box Transcription Factors

Interactions between proteins are essential for their functioning and the biological processes they control. The elucidation of interaction maps based on yeast studies is a first step toward the understanding of molecular networks and provides a framework of proteins that possess the capacity and specificity to interact. Here, we present a comprehensive plant protein–protein interactome map of nearly all members of the Arabidopsis thaliana MADS box transcription factor family. A matrix-based yeast two-hybrid screen of >100 members of this family revealed a collection of specific heterodimers and a few homodimers. Clustering of proteins with similar interaction patterns pinpoints proteins involved in the same developmental program and provides valuable information about the participation of uncharacterized proteins in these programs. Furthermore, a model is proposed that integrates the floral induction and floral organ formation networks based on the interactions between the proteins involved. Heterodimers between flower induction and floral organ identity proteins were observed, which point to (auto)regulatory mechanisms that prevent the activity of flower induction proteins in the flower.

MADS-Box Protein Complexes Control Carpel and Ovule Development in Arabidopsis

The AGAMOUS (AG) gene is necessary for stamen and carpel development and is part of a monophyletic clade of MADS-box genes that also includes SHATTERPROOF1 (SHP1), SHP2, and SEEDSTICK (STK). Here, we show that ectopic expression of either the STK or SHP gene is sufficient to induce the transformation of sepals into carpeloid organs bearing ovules. Moreover, the fact that these organ transformations occur when the STK gene is expressed ectopically in ag mutants shows that STK can promote carpel development in the absence of AG activity. We also show that STK, AG, SHP1, and SHP2 can form multimeric complexes and that these interactions require the SEPALLATA (SEP) MADS-box proteins. We provide genetic evidence for this role of the SEP proteins by showing that a reduction in SEP activity leads to the loss of normal ovule development, similar to what occurs in stk shp1 shp2 triple mutants. Together, these results indicate that the SEP proteins, which are known to form multimeric complexes in the control of flower organ identity, also form complexes to control normal ovule development.

Molecular control of ovule development

Enormous progress has been made over the past five years in understanding the floral homeotic genes, which specify the fate of the floral organ meristems. More recently, regulatory genes have been cloned from Petunia hybrida and Arabidopsis thaliana that play essential roles in the development of the ovule — the floral organ that contains the female gametophyte. Extensive mutagenesis programmes using Arabidopsis have yielded a number of ovule mutants that reflect the various steps in ovule ontogeny. These mutants, and the identification of the first key regulatory genes involved in ovule development, are powerful research tools for studies on plant reproduction. Moreover, ovule development is intriguing for evolutionary studies, because the ovule is a relatively simple organ found in both modern angiosperms and gymnosperms, and their common ancestor.

AGL24, SHORT VEGETATIVE PHASE, and APETALA1 Redundantly Control AGAMOUS during Early Stages of Flower Development in Arabidopsis

Loss-of-function alleles of AGAMOUS-LIKE24 (AGL24) and SHORT VEGETATIVE PHASE (SVP) revealed that these two similar MADS box genes have opposite functions in controlling the floral transition in Arabidopsis thaliana, with AGL24 functioning as a promoter and SVP as a repressor. AGL24 promotes inflorescence identity, and its expression is downregulated by APETALA1 (AP1) and LEAFY to establish floral meristem identity. Here, we combine the two mutants to generate the agl24 svp double mutant. Analysis of flowering time revealed that svp is epistatic to agl24. Furthermore, when grown at 30°C, the double mutant was severely affected in flower development. All four floral whorls showed homeotic conversions due to ectopic expression of class B and C organ identity genes. The observed phenotypes remarkably resembled the leunig (lug) and seuss (seu) mutants. Protein interaction studies showed that dimers composed of AP1-AGL24 and AP1-SVP interact with the LUG-SEU corepressor complex. We provide genetic evidence for the role of AP1 in these interactions by showing that the floral phenotype in the ap1 agl24 svp triple mutant is significantly enhanced. Our data suggest that MADS box proteins are involved in the recruitment of the SEU-LUG repressor complex for the regulation of AGAMOUS.

OsMADS13, a novel rice MADS-box gene expressed during ovule development.

MADS-box genes have been shown to play a major role in defining plant architecture. Recently, several MADS-box genes have been reported that are highly expressed in the ovule. However, only for the Petunia genes FBP7 and FBP11 has a function in defining ovule identity been shown. We have isolated a rice MADS-box gene named OsMADS13. Expression analysis has shown that this gene is highly expressed in developing ovules. In order to facilitate a detailed characterization of rice ovule-expressed genes, a comprehensive morphological description of ovule development in rice has been performed. The predicted amino acid sequence of OsMADS13 shows significant homology with ZAG2, a maize MADS-box gene, which is also expressed mainly in the ovule. Mapping of the gene in the rice genome showed that it is located on chromosome 12, which is syntenic to two maize regions where ZAG2 and its paralogous gene ZMM1 have been mapped. Our results suggest that OsMADS13 is the ortholog of ZAG2 and ZMM1 and might play a role in rice ovule and seed development.

Functional Characterization of OsMADS18, a Member of the AP1/SQUA Subfamily of MADS Box Genes

MADS box transcription factors controlling flower development have been isolated and studied in a wide variety of organisms. These studies have shown that homologous MADS box genes from different species often have similar functions. OsMADS18 from rice (Oryza sativa) belongs to the phylogenetically defined AP1/SQUA group. The MADS box genes of this group have functions in plant development, like controlling the transition from vegetative to reproductive growth, determination of floral organ identity, and regulation of fruit maturation. In this paper we report the functional analysis of OsMADS18. This rice MADS box gene is widely expressed in rice with its transcripts accumulated to higher levels in meristems. Overexpression of OsMADS18 in rice induced early flowering, and detailed histological analysis revealed that the formation of axillary shoot meristems was accelerated. Silencing of OsMADS18 using an RNA interference approach did not result in any visible phenotypic alteration, indicating that OsMADS18 is probably redundant with other MADS box transcription factors. Surprisingly, overexpression of OsMADS18 in Arabidopsis caused a phenotype closely resembling the ap1 mutant. We show that the ap1 phenotype is not caused by down-regulation of AP1 expression. Yeast two-hybrid experiments showed that some of the natural partners of AP1 interact with OsMADS18, suggesting that the OsMADS18 overexpression phenotype in Arabidopsis is likely to be due to the subtraction of AP1 partners from active transcription complexes. Thus, when compared to AP1, OsMADS18 during evolution seems to have conserved the mechanistic properties of protein-protein interactions, although it cannot complement the AP1 function.

Genetic and Molecular Interactions between BELL1 and MADS Box Factors Support Ovule Development in Arabidopsis

In Arabidopsis thaliana and many other plant species, ovules arise from carpel tissue as new meristematic formations. Cell fate in proliferating ovule primordia is specified by particular ovule identity factors, such as the homeodomain factor BELL1 (BEL1) and MADS box family members SEEDSTICK (STK), SHATTERPROOF1 (SHP1), SHP2, and AGAMOUS. Both in the bel1 mutant and the stk shp1 shp2 triple mutant, integuments are transformed into carpelloid structures. Combining these mutants in a bel1 stk shp1 shp2 quadruple mutant, we showed that the bel1 phenotype is significantly enhanced. We also demonstrate that ovule differentiation requires the regulation of the stem cell maintenance gene WUSCHEL, repression of which is predominantly maintained by BEL1 during ovule development. Based on yeast three-hybrid assays and genetic data, we show that BEL1 interacts with the ovule identity MADS box factors when they dimerize with SEPALLATA proteins. We propose a model for ovule development that explains how the balance between carpel identity activity and ovule identity activity is established by a MADS box homeodomain protein complex.

AGL23, A TYPE I MADS-BOX GENE THAT CONTROLS FEMALE GAMETOPHYTE AND EMBRYO DEVELOPMENT IN ARABIDOPSIS

MADS-box transcription factors are key regulators of plant developmental processes. While the function of MIKC (type II) MADS-box genes has been intensively studied, only limited data are available for the other more recently identified classes of MADS-box genes, despite these latter comprising more than 60% of the Arabidopsis MADS-box gene family. Here we describe the function of AGL23, an Arabidopsis type I MADS-box gene belonging to the Malpha subfamily. We show that AGL23 plays an important role during development of the female gametophyte and embryo. The agl23-1 mutant forms a functional megaspore. However, at this stage female gametophyte development is arrested and the megaspore persists during subsequent phases of ovule development. Despite the incomplete penetrance of the female gametophyte defect, plants homozygous for the agl23-1 mutation were never identified. Analysis of developing seeds showed that embryos homozygous for the agl23-1 allele are albino and unable to give rise to viable plants. Electron microscopy analysis revealed that this phenotype is due to the absence of chloroplasts, strongly suggesting that AGL23 is involved in controlling the biogenesis of organelles during embryo development.

Sex Determination in the Monoecious Species Cucumber Is Confined to Specific Floral Whorls

In unisexual flowers, sex is determined by the selective repression of growth or the abortion of either male or female reproductive organs. The mechanism by which this process is controlled in plants is still poorly understood. Because it is known that the identity of reproductive organs in plants is controlled by homeotic genes belonging to the MADS box gene family, we analyzed floral homeotic mutants from cucumber, a species that bears both male and female flowers on the same individual. To study the characteristics of sex determination in more detail, we produced mutants similar to class A and C homeotic mutants from well-characterized hermaphrodite species such as Arabidopsis by ectopically expressing and suppressing the cucumber gene CUCUMBER MADS1 (CUM1). The cucumber mutant green petals (gp) corresponds to the previously characterized B mutants from several species and appeared to be caused by a deletion of 15 amino acid residues in the coding region of the class B MADS box gene CUM26. These homeotic mutants reveal two important concepts that govern sex determination in cucumber. First, the arrest of either male or female organ development is dependent on their positions in the flower and is not associated with their sexual identity. Second, the data presented here strongly suggest that the class C homeotic function is required for the position-dependent arrest of reproductive organs.