Ovule-specific MADS-box proteins have conserved protein-protein interactions in monocot and dicot plants

OsMADS13 is a rice MADS-box gene that is specifically expressed in developing ovules. The amino acid sequence of OsMADS13 shows 74% similarity to those of FLORAL BINDING PROTEIN 7 (FBP7) and FBP11, the products of two MADS-box genes that are necessary and sufficient to determine ovule identity in Petunia. To assess whether OsMADS13, the putative rice ortholog of FBP7 and FBP11, has an equivalent function, several analyses were performed. Ectopic expression of FBP7 and FBP11 in Petunia results in ectopic ovule formation on sepals and petals. Here we show that ectopic expression of OsMADS13 in rice and Arabidopsis does not result in the formation of such structures. Furthermore, ectopic expression of FBP7 and FBP11 in Arabidopsis also fails to induce ectopic ovule formation. To determine whether protein-protein interactions involving putative class D MADS-box proteins have been conserved, yeast two-hybrid assays were performed. These experiments resulted in the identification of three putative partners of OsMADS13, all of them encoded by AGL2-like genes. Interestingly the Petunia FBP7 protein also interacts with AGL2-like proteins. The evolutionary conservation of the MADS-box protein partners of these ovule-specific factors was confirmed by exchange experiments which showed that the protein partners of OsMADS13 interact with FBP7 and vice versa.     

Members of TALE and WUS subfamilies of homeodomain proteins with potentially important functions in development form dimers within each subfamily in rice

Transacting factors often form homo- and heterodimers and regulate various targets, the type of regulation depending on the dimeric combination. The WUS and TALE subfamilies are two atypical homeodomains in plants. A homeodomain mediates sequence-specific binding to its target DNA and usually consists of 60 amino acid residues, whereas atypical homeodomains have extra amino acid residues in the well-conserved region. The genes OsWUS and OsPRS, which encode atypical homeodomain proteins from the WUS subfamily, and OsBEL and OSH15, which encode those from the TALE subfamily, were isolated from rice and tested for their interactions by yeast two-hybrid analysis. OsWUS and OsPRS formed homodimers and formed heterodimers with each other but did not form dimers with the TALE family homeodomain proteins OSH15 or OsBEL. Likewise, OSH15 and OsBEL formed homodimers and heterodimers but did not form dimers with the WUS family homeodomain proteins OsWUS and OsPRS. These findings suggest that the combinations of dimers are well correlated with the classification of these proteins on the basis of sequence similarity. RT-PCR analysis revealed that expression of OsWUS and OsPRS was detected in the same organs, namely floral buds, roots, and suspension cells. Therefore, it is possible that the proteins encoded by both of these genes function as homo- and heterodimers in planta. These results suggest that, during the evolution of these subfamilies, various combinations of dimers within proteins encoded by paralogous genes were formed and generated independent regulatory networks that enabled complex patterns of plant development.     

MADS-box转录因子的相互作用及对果实发育和成熟的调控

MADS-box基因编码的蛋白是一类数目庞大的转录因子家族, 通过与其他转录因子相互作用形成同源或异源二聚体, 调控着整个植株的生长发育。文章对近年来MADS-box转录因子的相互作用及对果实发育和成熟的调控作用的研究进展进行了综述, 为了解MADS-box转录因子的作用方式和作用机理及深入研究MADS-box基因对调控果实发育和成熟的作用提供参考。     

茶树花发育MADS-box转录因子CsGLO1、CsGLO2与CsAG之间的互作关系研究

利用酵母双杂交方法和双分子荧光互补技术（BiFC）研究了茶树（Camellia sinensis（L.）O.Kuntze）花发育MADS-box的B类转录因子CsGLO1、CsGLO2与C类转录因子CsAG间的互作关系及其可能发生互作的亚细胞定位。通过构建5个酵母表达载体,利用酵母单杂交实验检测了3个蛋白的转录激活活性,并通过酵母双杂交实验分析了蛋白间的互作关系。结果显示,3个蛋白均无转录激活活性,且两两之间可发生相互作用。进一步构建6个BiFC表达载体,采用压力注射法瞬时浸染烟草（Nicotiana benthamiana L.）叶表皮细胞,并利用激光共聚焦显微镜观察荧光信号,结果表明茶树B类CsGLO与C类CsAG蛋白可在植物活细胞内形成同源和异源二聚体,并具有在细胞质中发生互作的特定模式。本研究可为利用分子生物学技术抑制茶树“花果同现”现象提供理论依据。     

Structure-Function Analysis of Peroxisomal ATP-binding Cassette Transporters Using Chimeric Dimers

ABCD1 and ABCD2 are two closely related ATP-binding cassette half-transporters predicted to homodimerize and form peroxisomal importers for fatty acyl-CoAs. Available evidence has shown that ABCD1 and ABCD2 display a distinct but overlapping substrate specificity, although much remains to be learned in this respect as well as in their capability to form functional heterodimers. Using a cell model expressing an ABCD2-EGFP fusion protein, we first demonstrated by proximity ligation assay and co-immunoprecipitation assay that ABCD1 interacts with ABCD2. Next, we tested in the pxa1/pxa2Δ yeast mutant the functionality of ABCD1/ABCD2 dimers by expressing chimeric proteins mimicking homo- or heterodimers. For further structure-function analysis of ABCD1/ABCD2 dimers, we expressed chimeric dimers fused to enhanced GFP in human skin fibroblasts of X-linked adrenoleukodystrophy patients. These cells are devoid of ABCD1 and accumulate very long-chain fatty acids (C26:0 and C26:1). We checked that the chimeric proteins were correctly expressed and targeted to the peroxisomes. Very long-chain fatty acid levels were partially restored in transfected X-linked adrenoleukodystrophy fibroblasts regardless of the chimeric construct used, thus demonstrating functionality of both homo- and heterodimers. Interestingly, the level of C24:6 n-3, the immediate precursor of docosahexaenoic acid, was decreased in cells expressing chimeric proteins containing at least one ABCD2 moiety. Our data demonstrate for the first time that both homo- and heterodimers of ABCD1 and ABCD2 are functionally active. Interestingly, the role of ABCD2 (in homo- and heterodimeric forms) in the metabolism of polyunsaturated fatty acids is clearly evidenced, and the chimeric dimers provide a novel tool to study substrate specificity of peroxisomal ATP-binding cassette transporters.