A network of redundant bHLH proteins functions in all TTG1-dependent pathways of Arabidopsis

GLABRA3 (GL3) encodes a bHLH protein that interacts with the WD repeat protein, TTG1. GL3 overexpression suppresses the trichome defect of the pleiotropic ttg1 mutations. However, single gl3 mutations only affect the trichome pathway with a modest trichome number reduction. A novel unlinked bHLH-encoding locus is described here, ENHANCER OF GLABRA3 (EGL3). When mutated, egl3 gives totally glabrous plants only in the gl3 mutant background. The double bHLH mutant, gl3 egl3, has a pleiotropic phenotype like ttg1 having defective anthocyanin production, seed coat mucilage production, and position-dependent root hair spacing. Furthermore, the triple bHLH mutant, gl3 egl3 tt8, phenocopies the ttg1 mutation. Yeast two-hybrid and plant overexpression studies show that EGL3, like GL3, interacts with TTG1, the myb proteins GL1, PAP1 and 2, CPC and TRY, and it will form heterodimers with GL3. These results suggest a combinatorial model for TTG1-dependent pathway regulation by this trio of partially functionally redundant bHLH proteins.     

A mutation in the pale aleurone color1 gene identifies a novel regulator of the maize anthocyanin pathway.

By screening for new seed color mutations, we have identified a new gene, pale aleurone color1 (pac1), which when mutated causes a reduction in anthocyanin pigmentation. The pac1 gene is not allelic to any known anthocyanin biosynthetic or regulatory gene. The pac1-ref allele is recessive, nonlethal, and only reduces pigment in kernels, not in vegetative tissues. Genetic and molecular evidence shows that the pac1-ref allele reduces pigmentation by reducing RNA levels of the biosynthetic genes in the pathway. The mutant does not reduce the RNA levels of either of the two regulatory genes, b and c1. Introduction of an anthocyanin structural gene promoter (a1) driving a reporter gene into maize aleurones shows that pac1-ref kernels have reduced expression resulting from the action of the a1 promoter. Introduction of the reporter gene with constructs that express the regulatory genes b and c1 or the phlobaphene pathway regulator p shows that this reduction in a1-driven expression occurs in both the presence and absence of these regulators. Our results imply that pac1 is required for either b/c1 or p activation of anthocyanin biosynthetic gene expression and that pac1 acts independently of these regulatory genes.     

Transparent Testa Glabra 1 (TTG1) and TTG1-like genes in Matthiola incana R. Br. and related Brassicaceae and mutation in the WD-40 motif

TTG1 (Transparent Testa Glabra 1), a WD-40 repeat protein, is involved in regulation of flavonoid/anthocyanin biosynthesis, seed coat (mucilage) development/pigmentation and trichome formation in leaves. Here, we characterized the TTG1 gene of Matthiola incana wild type ( e locus), showing 85.3% similarity to TTG1 of A. thaliana on the nucleotide level and 96.2% on the protein level. A white-flowered and glabrous mutant, line 17, of M. incana exhibits one nucleotide change, leading to an amino acid substitution directly in the WD motif (W158R). Correspondingly, the DFR (dihydroflavonol 4-reductase) gene, in which the expression is known to be dependent on TTG1, is not expressed in Matthiola mutant lines 17 (and 19). Comparison of the GC content of the Matthiola TTG1 (54.1%) and Arabidopsis TTG1 (46.1%) genes revealed a strong difference, mostly obtained by neutral substitutions (C to T transitions). To examine whether this is an ecologically influenced trend, a fragment of TTG1 was characterized from another Matthiola species ( M. tricuspidata ) and from Malcolmia flexuosa subsp. naxensis from the eastern Mediterranean, near a beach with sandy and salty soils. Both Matthiola species have a higher GC content in the TTG1 gene than Arabidopsis and the closer-related Malcolmia , indicating that the GC content is rather an evolutionary than an ecological signal. A similar WD-40 repeat protein gene (containing no intron in the 3' untranslated region) with high similarity to the Arabidopsis TTG1 -like ( AtAN11 ) gene was found in Matthiola .     

WD40 Domain Divergence Is Important for Functional Differences between the Fission Yeast Tup11 and Tup12 Co-Repressor Proteins

We have previously demonstrated that subsets of Ssn6/Tup target genes have distinct requirements for the Schizosaccharomyces pombe homologs of the Tup1/Groucho/TLE co-repressor proteins, Tup11 and Tup12. The very high level of divergence in the histone interacting repression domains of the two proteins suggested that determinants distinguishing Tup11 and Tup12 might be located in this domain. Here we have combined phylogenetic and structural analysis as well as phenotypic characterization, under stress conditions that specifically require Tup12, to identify and characterize the domains involved in Tup12-specific action. The results indicate that divergence in the repression domain is not generally relevant for Tup12-specific function. Instead, we show that the more highly conserved C-terminal WD40 repeat domain of Tup12 is important for Tup12-specific function. Surface amino acid residues specific for the WD40 repeat domain of Tup12 proteins in different fission yeasts are clustered in blade 3 of the propeller-like structure that is characteristic of WD40 repeat domains. The Tup11 and Tup12 proteins in fission yeasts thus provide an excellent model system for studying the functional divergence of WD40 repeat domains.     

Subcellular localization and functional domain studies of DEFECTIVE KERNEL1 in maize and Arabidopsis suggest a model for aleurone cell fate specification involving CRINKLY4 and SUPERNUMERARY ALEURONE LAYER1

DEFECTIVE KERNEL1 (DEK1), which consists of a membrane-spanning region (DEK1-MEM) and a calpain-like Cys proteinase region (DEK1-CALP), is essential for aleurone cell formation at the surface of maize (Zea mays) endosperm. Immunolocalization and FM4-64 dye incubation experiments showed that DEK1 and CRINKLY4 (CR4), a receptor kinase implicated in aleurone cell fate specification, colocalized to plasma membrane and endosomes. SUPERNUMERARY ALEURONE LAYER1 (SAL1), a negative regulator of aleurone cell fate encoding a class E vacuolar sorting protein, colocalized with DEK1 and CR4 in endosomes. Immunogold localization, dual-axis electron tomography, and diffusion of fluorescent dye tracers showed that young aleurone cells established symplastic subdomains through plasmodesmata of larger dimensions than those connecting starchy endosperm cells and that CR4 preferentially associated with plasmodesmata between aleurone cells. Genetic complementation experiments showed that DEK1-CALP failed to restore wild-type phenotypes in maize and Arabidopsis thaliana dek1 mutants, and DEK1-MEM also failed to restore wild-type phenotypes in Arabidopsis dek1-1 mutants. Instead, ectopic expression of DEK1-MEM under the control of the cauliflower mosaic virus 35S promoter gave a dominant negative phenotype. These data suggest a model for aleurone cell fate specification in which DEK1 perceives and/or transmits a positional signal, CR4 promotes the lateral movement of aleurone signaling molecules between aleurone cells, and SAL1 maintains the proper plasma membrane concentration of DEK1 and CR4 proteins via endosome-mediated recycling/degradation.     

MSI1-like proteins: an escort service for chromatin assembly and remodeling complexes

MSI1-like WD40 repeat proteins are subunits of many protein complexes controlling chromatin dynamics. These proteins do not have any catalytic activity, but several recent studies using loss-of-function mutants established specific functions during development. Here, we review the current knowledge of MSI1-like proteins, including their phylogenetic history, expression patterns, biochemical interactions and mutant phenotypes. MSI1-like proteins, which are often targets or partners of tumor-suppressor proteins, are required during cell proliferation and differentiation in flies, nematodes and plants. We discuss the possibility that MSI1-like proteins could function to maintain epigenetic memory during development by targeting silencing complexes to chromatin during nucleosome assembly.     

甘蓝型油菜BnTTG1-1基因的功能分析

拟南芥(Arabidopsis thaliana)AtTTG1作为WD40重复转录因子存在于细胞核中,对表皮毛形成、花青素合成和储藏物质积累等具有重要调节作用。该研究从甘蓝型油菜(Brassica napus)品种秦优7号中克隆获得了BnTTG1-1基因的全长CDS序列,对其进行了烟草(Nicotiana benthamiana)叶片细胞的亚细胞定位研究,检测了BnTTG1-1在油菜(B.campestris)中的时空表达模式,并比较分析了BnTTG1-1对多个生物学过程的影响作用。结果表明,BnTTG1-1定位于烟草叶片细胞的细胞核中,推测其作为转录因子发挥调节作用。BnTTG1-1广泛存在于油菜营养组织和发育的种子中。在突变体ttg1-13背景下,异源表达BnTTG1-1基因能够完全恢复该突变体的多个表型,如无表皮毛形成和花青素合成、种皮呈黄色、种子脂肪酸和储藏蛋白含量高以及在种子萌发和幼苗形态建成过程中对高葡萄糖和高盐胁迫耐受力差等。由此可知,甘蓝型油菜BnTTG1-1与拟南芥AtTTG1在植物生长发育的多个生物学过程中具有类似的功能。     

植物色氨酸一天冬氨酸重复序列蛋白响应逆境胁迫的调控作用

干旱、盐渍、低温等逆境胁迫会严重影响植物的正常生长发育,导致植物的许多响应基因被诱导表达,其蛋白质产物能够保护植物免受胁迫的伤害。色氨酸一天冬氨酸重复序列蛋白（wD40蛋自）在植物中广泛存在,参与植物体内众多代谢反应的调控,如花的发育、开花、花青素的生物合成、激素响应、渗透胁迫等。WD40蛋白含有40-60个氨基酸的保守的wD重复序列,其c末端为色氨酸．天冬氨酸（Trp-Asp,WD）,形成一个p螺旋桨（p—propeller）结构,通过调节多蛋白复合体的组装而影响蛋白质与蛋白质、蛋白质与DNA间的相互作用。本文综述植物WD40蛋白响应逆境胁迫的调控作用。     

High expression of the penicillin G acylase gene (pac) from Bacillus megaterium UN1 in its own pac minus mutant

By marker exchange mutagenesis, Bacillus megaterium strain UN-1 (Bm-UN1) was used to prepare a mutant strain B. megaterium UN-cat (Bm-UNcat) lacking the penicillin G acylase gene (pac). The pac gene from Bm-UN1 was subcloned into pTF6 and the resultant plasmid, pBA402, was introduced into Bm-UNcat and Bacillus subtilis. Bm-UNcat harbouring pBA402 produced high penicillin G acylase (PAC) activity of 13.7, 19.5 and 20.4 U ml(-1) at 24, 36 and 48 h of culture, respectively. This was two- to fivefold higher than PAC produced by B. subtilis harbouring pBA402 and about 20-fold higher than PAC produced by the parent strain, Bm-UN1.     

A WD40 repeat protein regulates fungal cell differentiation and can be replaced functionally by the mammalian homologue striatin

Fruiting body development in fungi is a complex cellular differentiation process that is controlled by more than 100 developmental genes. Mutants of the filamentous fungus Sordaria macrospora showing defects in fruiting body formation are pertinent sources for the identification of components of this multicellular differentiation process. Here we show that the sterile mutant pro11 carries a defect in the pro11 gene encoding a multimodular WD40 repeat protein. Complementation analysis indicates that the wild-type gene or C-terminally truncated versions of the wild-type protein are able to restore the fertile phenotype in mutant pro11. PRO11 shows significant homology to several vertebrate WD40 proteins, such as striatin and zinedin, which seem to be involved in Ca²⁺-dependent signaling in cells of the central nervous system and are supposed to function as scaffolding proteins linking signaling and eukaryotic endocytosis. Cloning of a mouse cDNA encoding striatin allowed functional substitution of the wild-type protein with restoration of fertility in mutant pro11. Our data strongly suggest that an evolutionarily conserved cellular process controlling eukaryotic cell differentiation may regulate fruiting body formation.     

The Molecular Chaperone TRiC/CCT Binds to the Trp-Asp 40 (WD40) Repeat Protein WDR68 and Promotes Its Folding,Protein Kinase DYRK1A Binding,and Nuclear Accumulation

Trp-Asp (WD) repeat protein 68 (WDR68) is an evolutionarily conserved WD40 repeat protein that binds to several proteins, including dual specificity tyrosine phosphorylation-regulated protein kinase (DYRK1A), MAPK/ERK kinase kinase 1 (MEKK1), and Cullin4-damage-specific DNA-binding protein 1 (CUL4-DDB1). WDR68 affects multiple and diverse physiological functions, such as controlling anthocyanin synthesis in plants, tissue growth in insects, and craniofacial development in vertebrates. However, the biochemical basis and the regulatory mechanism of WDR68 activity remain largely unknown. To better understand the cellular function of WDR68, here we have isolated and identified cellular WDR68 binding partners using a phosphoproteomic approach. More than 200 cellular proteins with wide varieties of biochemical functions were identified as WDR68-binding protein candidates. Eight T-complex protein 1 (TCP1) subunits comprising the molecular chaperone TCP1 ring complex/chaperonin-containing TCP1 (TRiC/CCT) were identified as major WDR68-binding proteins, and phosphorylation sites in both WDR68 and TRiC/CCT were identified. Co-immunoprecipitation experiments confirmed the binding between TRiC/CCT and WDR68. Computer-aided structural analysis suggested that WDR68 forms a seven-bladed β-propeller ring. Experiments with a series of deletion mutants in combination with the structural modeling showed that three of the seven β-propeller blades of WDR68 are essential and sufficient for TRiC/CCT binding. Knockdown of cellular TRiC/CCT by siRNA caused an abnormal WDR68 structure and led to reduction of its DYRK1A-binding activity. Concomitantly, nuclear accumulation of WDR68 was suppressed by the knockdown of TRiC/CCT, and WDR68 formed cellular aggregates when overexpressed in the TRiC/CCT-deficient cells. Altogether, our results demonstrate that the molecular chaperone TRiC/CCT is essential for correct protein folding, DYRK1A binding, and nuclear accumulation of WDR68.