RESUMEN
The MADS transcription factors (TF) are an ancient eukaryotic protein family. In plants, the family is divided into two main lineages. Here, we demonstrate that DNA binding in both lineages absolutely requires a short amino acid sequence C-terminal to the MADS domain (M domain) called the Intervening domain (I domain) that was previously defined only in type II lineage MADS. Structural elucidation of the MI domains from the floral regulator, SEPALLATA3 (SEP3), shows a conserved fold with the I domain acting to stabilise the M domain. Using the floral organ identity MADS TFs, SEP3, APETALA1 (AP1) and AGAMOUS (AG), domain swapping demonstrate that the I domain alters genome-wide DNA-binding specificity and dimerisation specificity. Introducing AG carrying the I domain of AP1 in the Arabidopsis ap1 mutant resulted in strong complementation and restoration of first and second whorl organs. Taken together, these data demonstrate that the I domain acts as an integral part of the DNA-binding domain and significantly contributes to the functional identity of the MADS TF.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Homeodominio/química , Factores de Transcripción/química , Proteína AGAMOUS de Arabidopsis/química , Proteína AGAMOUS de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Unión al ADN/metabolismo , Flores , Regulación de la Expresión Génica de las Plantas , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas de Dominio MADS/metabolismo , Fenotipo , Dominios y Motivos de Interacción de Proteínas , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The carpel- and stamen-specific AtAGIP promoter derived from the Arabidopsis AGAMOUS (AG) second intron/enhancer is ideal for engineering complete sterility but it is highly host-specific. To ascertain whether a chimeric promoter with similar tissue specificity can be created for species other than Arabidopsis, we isolated two similar but distinct AG second intron/enhancers from tobacco (NtAGI-1 and NtAGI-2) and analyzed their ability to drive floral organ-specific expression in plants through the creation of forward- and reverse-oriented chimeric promoters, fNtAGIP1, rNtAGIP1, fNtAGIP2 and rNtAGIP2. Analyses of transgenic plants bearing each respective promoter fused to the beta-glucuronidase (GUS) reporter gene showed that all four promoters are able, like the AtAGIP, to drive very similar carpel- and stamen-specific expression without any leaky activity in vegetative tissues. These results indicate that unlike their counterparts in rice and maize, the tobacco NtAGI-1 and NtAGI-2 enhancers share a highly conserved regulatory function. Interestingly, all four promoters display additional tissue specificity in petals, and their activity is influenced by the orientation of the incorporated enhancer, with reverse-oriented enhancers exhibiting approximately double the effectiveness of forward-oriented enhancers. These properties are novel and have not been observed with the AtAGIP promoter in Arabidopsis. As expected, these highly specific promoters can also direct the expression of the DT-A cytotoxic gene exclusively in carpels, stamens and petals, resulting in complete sterility through the precise ablation of targeted floral organs. Further analyses demonstrated that the resulting trait is mitotically stable, which is critical for the long-term containment of seed-, pollen- and fruit-mediated gene flow in field conditions.
Asunto(s)
Proteína AGAMOUS de Arabidopsis/química , Flores/genética , Regulación de la Expresión Génica de las Plantas , Intrones/genética , Nicotiana/genética , Infertilidad Vegetal/genética , Proteínas de Plantas/genética , Clonación Molecular , Elementos de Facilitación Genéticos/genética , Flores/citología , Ingeniería Genética , Genoma de Planta/genética , Glucuronidasa/metabolismo , Mitosis , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Homología de Secuencia de Aminoácido , Nicotiana/citologíaRESUMEN
MADS box genes in plants consist of MIKC-type and type I genes. While MIKC-type genes have been studied extensively, the functions of type I genes are still poorly understood. Evidence suggests that type I MADS box genes are involved in embryo sac and seed development. We investigated two independent T-DNA insertion alleles of the Arabidopsis thaliana type I MADS box gene AGAMOUS-LIKE61 (AGL61) and showed that in agl61 mutant ovules, the polar nuclei do not fuse and central cell morphology is aberrant. Furthermore, the central cell begins to degenerate before fertilization takes place. Although pollen tubes are attracted and perceived by the mutant ovules, neither endosperm development nor zygote formation occurs. AGL61 is expressed in the central cell during the final stages of embryo sac development. An AGL61:green fluorescent protein-beta-glucoronidase fusion protein localizes exclusively to the polar nuclei and the secondary nucleus of the central cell. Yeast two-hybrid analysis showed that AGL61 can form a heterodimer with AGL80 and that the nuclear localization of AGL61 is lost in the agl80 mutant. Thus, AGL61 and AGL80 appear to function together to differentiate the central cell in Arabidopsis. We renamed AGL61 DIANA, after the virginal Roman goddess of the hunt.
Asunto(s)
Proteína AGAMOUS de Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Flores/metabolismo , Proteínas de Dominio MADS/metabolismo , Proteína AGAMOUS de Arabidopsis/química , Proteína AGAMOUS de Arabidopsis/genética , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Dimerización , Flores/genética , Flores/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Proteínas de Dominio MADS/química , Proteínas de Dominio MADS/genética , Datos de Secuencia Molecular , Fenotipo , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Técnicas del Sistema de Dos HíbridosRESUMEN
In Impatiens balsamina a lack of commitment of the meristem during floral development leads to the continuous requirement for a leaf-derived floral signal. In the absence of this signal the meristem reverts to leaf production. Current models for Arabidopsis state that LEAFY (LFY) is central to the integration of floral signals and regulates flowering partly via interactions with TERMINAL FLOWER1 (TFL1) and AGAMOUS (AG). Here we describe Impatiens homologues of LFY, TFL1 and AG (IbLFY, IbTFL1 and IbAG) that are highly conserved at a sequence level and demonstrate homologous functions when expressed ectopically in transgenic Arabidopsis. We relate the expression patterns of IbTFL1 and IbAG to the control of terminal flowering and floral determinacy in Impatiens. IbTFL1 is involved in controlling the phase of the axillary meristems and is expressed in axillary shoots and axillary meristems which produce inflorescences, but not in axillary flowers. It is not involved in maintaining the terminal meristem in either an inflorescence or indeterminate state. Terminal flowering in Impatiens appears therefore to be controlled by a pathway that uses a different integration system than that regulating the development of axillary flowers and branches. The pattern of ovule production in Impatiens requires the meristem to be maintained after the production of carpels. Consistent with this morphological feature IbAG appears to specify stamen and carpel identity, but is not sufficient to specify meristem determinacy in Impatiens.
Asunto(s)
Flores/crecimiento & desarrollo , Impatiens/crecimiento & desarrollo , Proteínas de Plantas/química , Proteínas de Plantas/fisiología , Proteína AGAMOUS de Arabidopsis/química , Secuencia de Aminoácidos , Arabidopsis/anatomía & histología , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Diferenciación Celular/genética , Clonación Molecular , Flores/anatomía & histología , Flores/metabolismo , Regulación de la Expresión Génica de las Plantas , Impatiens/anatomía & histología , Impatiens/genética , Meristema/genética , Meristema/metabolismo , Datos de Secuencia Molecular , Filogenia , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/anatomía & histología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Transcripción/químicaRESUMEN
Floral organs, whose identity is determined by specific combinations of homeotic genes, originate from a group of undifferentiated cells called the floral meristem. In Arabidopsis, the homeotic gene AGAMOUS (AG) terminates meristem activity and promotes development of stamens and carpels. To understand the program of gene expression activated by AG, we followed genome-wide expression during early stamen and carpel development. The AG target genes included most genes for which mutant screens revealed a function downstream of AG. Novel targets were validated by in situ hybridisation and binding to AG in vitro and in vivo. Transcription factors formed a large fraction of AG targets, suggesting that during early organogenesis, much of the genetic program is concerned with elaborating gene expression patterns. The results also suggest that AG and other homeotic proteins with which it interacts (SEPALLATA3, APETALA3, PISTILLATA) are coordinately regulated in a positive-feedback loop to maintain their own expression, and that AG activates biosynthesis of gibberellin, which has been proposed to promote the shift from meristem identity to differentiation.
Asunto(s)
Proteína AGAMOUS de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , Flores/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes Homeobox/genética , Transcripción Genética/genética , Proteína AGAMOUS de Arabidopsis/química , Proteína AGAMOUS de Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Secuencia de Bases , Flores/crecimiento & desarrollo , Proteínas de Homeodominio/metabolismo , Hibridación in Situ , Proteínas de Dominio MADS/química , Proteínas de Dominio MADS/genética , Proteínas de Dominio MADS/metabolismo , Mutación/genética , Proteínas de Plantas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Factores de TiempoRESUMEN
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.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , Genes de Plantas/genética , Proteínas de Dominio MADS/genética , Proteínas de Dominio MADS/metabolismo , Estructuras de las Plantas/crecimiento & desarrollo , Estructuras de las Plantas/genética , Proteína AGAMOUS de Arabidopsis/química , Proteína AGAMOUS de Arabidopsis/genética , Proteína AGAMOUS de Arabidopsis/metabolismo , Alelos , Arabidopsis/anatomía & histología , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Frutas/genética , Frutas/crecimiento & desarrollo , Proteínas de Dominio MADS/química , Microscopía Electrónica de Rastreo , Morfogénesis , Semillas/genética , Semillas/crecimiento & desarrolloRESUMEN
We are interested in identifying potential protein interactors of MADS domain transcription factors during Arabidopsis thaliana flower development. We based our biochemical search on a conserved motif in the MADS domain that includes putative phosphatase and phosphorylation sites that may mediate protein interactions. An affinity column with this motif and a few surrounding hypervariable amino acids derived from the AGAMOUS sequence was prepared and used to isolate potential interactors from floral crude extracts. Only two proteins were specifically bound to the affinity column. The first corresponds to a carpel specific storage protein, VSP1, that presents acid phosphatase activity, and the second is a novel leucine-rich repeat protein that we have named FLOR1. Coimmunoprecipitation, two-hybrid yeast, and affinity column assays show that the FLOR1-VSP1 complex interacts with AGAMOUS and that this transcription factor directly interacts with FLOR1. This is the first assay to show an interaction between plant MADS domain factors and non-MADS proteins.