The K domain mediates heterodimerization of the Arabidopsis floral organ identity proteins,APETALA3 and PISTILLATA

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.     

Determination of floral organ identity by Arabidopsis MADS domain homeotic proteins AP1, AP3, PI, and AG is independent of their DNA-binding specificity.

The MADS domain homeotic proteins APETALA1 (AP1), APETALA3 (AP3), PISTILLATA (PI), and AGAMOUS (AG) combinatorially specify the identity of Arabidopsis floral organs. AP1/AP1, AG/AG, and AP3/PI dimers bind to similar CArG box sequences; thus, differences in DNA-binding specificity among these proteins do not seem to be the origin of their distinct organ identity properties. To assess the overall contribution that specific DNA binding could make to their biological specificity, we have generated chimeric genes in which the amino-terminal half of the MADS domain of AP1, AP3, PI, and AG was substituted by the corresponding sequences of human SRF and MEF2A proteins. In vitro DNA-binding assays reveal that the chimeric proteins acquired the respective, and distinct, DNA-binding specificity of SRF or MEF2A. However, ectopic expression of the chimeric genes reproduces the dominant gain-of-function phenotypes exhibited by plants ectopically expressing the corresponding Arabidopsis wild-type genes. In addition, both the SRF and MEF2 chimeric genes can complement the pertinent ap1-1, ap3-3, pi-1, or ag-3 mutations to a degree similar to that of AP1, AP3, PI, and AG when expressed under the control of the same promoter. These results indicate that determination of floral organ identity by the MADS domain homeotic proteins AP1, AP3, PI, and AG is independent of their DNA-binding specificity. In addition, the DNA-binding experiments show that either one of the two MADS domains of a dimer can be sufficient to confer a particular DNA-binding specificity to the complex and that sequences outside the amino-terminal basic region of the MADS domain can, in some cases, contribute to the DNA-binding specificity of the proteins.     

Conserved C-terminal motifs of the Arabidopsis proteins APETALA3 and PISTILLATA are dispensable for floral organ identity function

The B-class genes APETALA3 (AP3) and PISTILLATA (PI) in Arabidopsis (Arabidopsis thaliana) and their orthologs in other species have been the focus of studies to elucidate the development of petals and stamens in angiosperm flowers. Evolutionary analysis indicates that B-class genes have undergone multiple gene duplication events in angiosperms. The resultant B-class lineages are characterized by short, conserved amino acid sequences at the extreme C-terminal end of the B-class proteins. AP3 is a member of the euAP3 lineage that contains both the euAP3 and PI-derived motifs at the C terminus. PI is a member of the PI lineage that contains the C-terminal PI motif at the C terminus. Despite conservation over a wide evolutionary distance, the function of C-terminal motifs is not well understood. In this study, we demonstrate that truncated forms of AP3 and PI, which lack the conserved C-terminal motifs, function to direct floral organ identity specification in Arabidopsis plants. By contrast, larger truncations, which remove the third putative amphipathic alpha-helix in the K domain of AP3 or PI, are nonfunctional. We conclude that the euAP3 and PI-derived motifs of AP3 and the PI motif of PI are not essential for floral organ identity function of AP3 and PI in Arabidopsis.     

Nucleotide sequences recognized by the AGAMOUS MADS domain of Arabidopsis thaliana in vitro

The AGAMOUS gene of Arabidopsis thaliana is a homeotic gene involved in the development of stamens and carpels. This gene encodes a putative DNA-binding protein sharing a homologous region with the DNA-binding domains, MADS boxes, of yeast MCM1 and mammalian SRF. To examine the DNA-binding activity of the AGAMOUS protein, double-stranded oligonucleotides with random sequences of 40 bp in the central region were synthesized and mixed with the AGAMOUS MADS domain overproduced in Escherichia coli . Oligonucleotides which bound to the MADS domain were recovered by repeated immunoprecipitation with an antibody which recognizes the overproduced protein. From a comparison of the recovered DNA sequences, the consensus sequence of the high-affinity binding-sites for the AGAMOUS MADS domain was determined to be 5'-TT(A/T/G) CC(A/T)₆GG(A/T/C)AA-3'. DNase I footprinting and methylation interference experiments showed that the MADS domain binds to this motif. Comparisons with the binding-site sequences of other MADS-box proteins revealed that the MCM1 binding-sites in a-mating type-specific promoters of Saccharomyces cerevisiae show similarities with the binding-site sequence of the AGAMOUS MADS domain. A synthetic MCM1 binding-site in the upstream region of the STE2 gene is recognized by the AGAMOUS MADS domain.     

Inhibitor of apoptosis (IAP)-like protein lacks a baculovirus IAP repeat (BIR) domain and attenuates cell death in plant and animal systems

A novel Arabidopsis thaliana inhibitor of apoptosis was identified by sequence homology to other known inhibitor of apoptosis (IAP) proteins. Arabidopsis IAP-like protein (AtILP) contained a C-terminal RING finger domain but lacked a baculovirus IAP repeat (BIR) domain, which is essential for anti-apoptotic activity in other IAP family members. The expression of AtILP in HeLa cells conferred resistance against tumor necrosis factor (TNF)-α/ActD-induced apoptosis through the inactivation of caspase activity. In contrast to the C-terminal RING domain of AtILP, which did not inhibit the activity of caspase-3, the N-terminal region, despite displaying no homology to known BIR domains, potently inhibited the activity of caspase-3 in vitro and blocked TNF-α/ActD-induced apoptosis. The anti-apoptotic activity of the AtILP N-terminal domain observed in plants was reproduced in an animal system. Transgenic Arabidopsis lines overexpressing AtILP exhibited anti-apoptotic activity when challenged with the fungal toxin fumonisin B1, an agent that induces apoptosis-like cell death in plants. In AtIPL transgenic plants, suppression of cell death was accompanied by inhibition of caspase activation and DNA fragmentation. Overexpression of AtILP also attenuated effector protein-induced cell death and increased the growth of an avirulent bacterial pathogen. The current results demonstrated the existence of a novel plant IAP-like protein that prevents caspase activation in Arabidopsis and showed that a plant anti-apoptosis gene functions similarly in plant and animal systems.     

DNA-binding properties of Arabidopsis MADS domain homeotic proteins APETALA1, APETALA3, PISTILLATA and AGAMOUS.

The MADS domain proteins APETALA1 (AP1), APETALA3 (AP3), PISTILLATA (PI), and AGAMOUS (AG) specify the identity of Arabidopsis floral organs. AP1 and AG homocomplexes and AP3-PI heterocomplexes bind to CArG-box sequences. The DNA-binding properties of these complexes were investigated. We find that AP1, AG and AP3-PI are all capable of recognizing the same DNA-binding sites, although with somewhat different affinities. In addition, the three complexes induce similar conformational changes on a CArG-box sequence. Phasing analysis reveals that the induced distortion is DNA bending, oriented toward the minor groove. The molecular dissection of AP1, AP3, PI and AG indicates that the boundaries of the dimerization domains of these proteins vary. The regions required to form a DNA-binding complex include, in addition to the MADS box, the entire L region (which follows the MADS box) and the first putative amphipathic helix of the K box in the case of AP3-PI, while for AP1 and AG only a part of the L region is needed. The similarity of the DNA-binding properties of AP1, AP3-PI and AG is discussed with regard to the biological specificity that these proteins exhibit.     

Defining subdomains of the K domain important for protein–protein interactions of plant MADS proteins

The MADS proteins APETALA3 (AP3), PISTILLATA (PI), SEPALLATAI (SEPI), SEP2, SEP3, AGAMOUS, and APETALA are required for proper floral organ identity in Arabidopsis flowers. All of these floral MADS proteins conserve two domains: the MADS domain that mediates DNA binding and dimerization, and the K domain that mediates protein protein interaction. The K domain is postulated to form a several amphipathic c-helices referred to as K1, K2, and K3. The K1 and K2 helicies are located entirely within the K domain while the K3 helix spans the K domain-C domain boundary. Here we report on our studies on the interactions of the B class MADS proteins AP3 and PI with the E class MADS proteins SEP1, SEP2, and SEP3. A comparative analysis of mutants in the K domain reveals that the subdomains mediating the PI/AP3 interaction are different from the subdomains mediating the PI/SEP3 (or PI/SEP1) interaction. The strong PI/SEP3 (or PI/SEP1) interaction requires K2, part of K3, and the interhelical region between K1 and K2. By contrast, K1, K2 and the region between K1 and K2 are important for strong AP3/PI interaction. Most of the K3 helix does not appear to be important for either the PI/AP3 or the PI/SEP3 (or PI/SEP1) interaction. Conserved hydrophobic positions are most important for the strength of both PI/AP3 and PI/SEP3 dimerization, though ionic and/or polar interactions appear to play a secondary role.     

与花球发生相关的BoCAL和BoAP1互作因子的筛选

CAL(CAULIFLOWER)基因与AP1(APETALA1)基因都是控制花分生组织发育的基因,二者都属于MADS-box转录因子编码基因,在拟南芥中,它们同时突变时会使花分生组织保持花序分生组织的无限分生特性,大量增生分生组织结构,形成花球表型。而花椰菜(Brasscia oleracea L.var.botrytis)中BobCAL基因单突变就能形成花球,显然两个物种中CAL的功能可能不同。为了研究芸苔属植物中CAL和AP1同源蛋白的功能,尤其是在花球形成方面的调控作用,我们利用酵母双杂交方法对拟南芥中结球甘蓝(B.oleracea vat capitata L.)BoCAL的互作因子进行了筛选。与BoCAL互作较强的四类蛋白,分别涉及蛋白质的磷酸化和去磷酸化、蛋白质的修饰、蛋白质的结合位点等,它们分别与转录调控途径及信号转导途径有着密切的联系,这些因子的获得为BoCAL作用机制研究提供了线索。我们同时检测了部分BoCAL的互作因子和BoAP1之间的互作关系以及部分已知的MADS盒转录因子分别与BoCAL和BoAP1的互作,结果表明BoCAL特异性地与SnRKβ2互作,BoCAL、BoAP1和拟南芥中同源蛋白都能与SVP互作,但与拟南芥中同源蛋白不同的是,BoCAL、BoAP1与FLM、SOC1(SUPPRESSOR OF CO OVEREXPRESSION 1)和AGL24(AGAMOUS-LIKE24)作用很弱或不能互作,暗示BoCAL和BoAP1与拟南芥中同源蛋白功能上是不同的。