Polycomb Group蛋白复合体与植物春化作用

Polycomb Group(PcG)蛋白能形成Polycomb Repressive Complex 1(PRC1)和PRC2等复合体,通过一个保守且表观遗传的机制调节基因表达并控制动植物的发育。拟南芥中由VERNALIZATION2参与形成的PRC2复合体(VRN2-PRC2)在春化过程中能对主要开花抑制基因FLOWER LOCUS C(FLC)的染色质进行组蛋白甲基化修饰,形成H3K27me3(组蛋白H3第27位赖氨酸三甲基化)等转录抑制标记,从而抑制FLC转录,促进开花。虽然麦类作物的春化机理与拟南芥有较大差异,但最近的研究表明麦类作物春化过程也受PcG蛋白调控。文章对拟南芥PcG蛋白介导的春化调节机制进行综述,期望能对植物尤其是麦类作物的春化机理研究提供资料。     

Cortical morphogenesis in Paramecium: a transcellular wave of protein phosphorylation involved in ciliary rootlet disassembly

In Paramecium, the morphogenesis of the cortex at cell division, which assures reconstruction of shape and surface pattern, has been shown to involve transcellular signals which spread across the cortex like a wave, originating principally from the oral apparatus. One of the events these signals control is the reorganization of the ciliary rootlets through a cycle of regression and regrowth. The ciliary rootlets are nucleated on the ciliary basal bodies and form a scaffold extending over the entire cell surface that is important in aligning the basal bodies and the unit territories organized around them in longitudinal rows. We present evidence that the mechanism underlying their reorganization is cell-cycle-dependent phosphorylation of the structural proteins which compose the ciliary rootlets. We have isolated the rootlets and prepared a polyclonal antibody against them. In situ immunofluorescence of dividing cells with the anti rootlet antibody, and with the monoclonal antibody MPM-2 specific for phosphoproteins shows that a wave of phosphorylation of the ciliary rootlets spreads across the cell at division and just precedes their regression. Two-dimensional Western blot analysis of cytoskeleton and isolated rootlets along with alkaline phosphatase treatment demonstrates that the rootlets are composed of phosphoproteins, while experiments with interphase and dividing cells provide direct evidence that hyperphosphorylation of these proteins at division brings about disassembly of the structure.     

The putative Arabidopsis arp2/3 complex controls leaf cell morphogenesis

The evolutionarily conserved Arp2/3 complex has been shown to activate actin nucleation and branching in several eukaryotes, but its biological functions are not well understood in multicellular organisms. The model plant Arabidopsis provides many advantages for genetic dissection of the function of this conserved actin-nucleating machinery, yet the existence of this complex in plants has not been determined. We have identified Arabidopsis genes encoding homologs of all of the seven Arp2/3 subunits. The function of the putative Arabidopsis Arp2/3 complex has been studied using four homozygous T-DNA insertion mutants for ARP2, ARP3, and ARPC5/p16. All four mutants display identical defects in the development of jigsaw-shaped epidermal pavement cells and branched trichomes in the leaf. These loss-of-function mutations cause mislocalization of diffuse cortical F-actin to the neck region and inhibit lobe extension in pavement cells. The mutant trichomes resemble those treated with the actin-depolymerizing drug cytochalasin D, exhibiting stunted branches but dramatically enlarged stalks due to depolarized growth suggesting defects in the formation of a fine actin network. Our data demonstrate that the putative Arabidopsis Arp2/3 complex controls cell morphogenesis through its roles in cell polarity establishment and polar cell expansion. Furthermore, our data suggest a novel function for the putative Arp2/3 complex in the modulation of the spatial distribution of cortical F-actin and provide evidence that the putative Arp2/3 complex may activate the polymerization of some types of actin filaments in specific cell types.     

A putative FAD-binding domain in a distinct group of oxidases including a protein involved in plant development.

Using methods for database screening with individual protein sequences and alignment blocks, a conserved domain is delineated in a group of proteins including several FAD-dependent oxidases. Two motifs within this domain resemble phosphate-binding loops and may be directly involved in FAD binding. These motifs can be readily distinguished from previously described nucleotide-binding sites using a method for database screening with position-dependent weight matrices derived from alignment blocks. Unexpectedly, this group of known and predicted FAD-dependent oxidases includes the product of the DIMINUTO gene, which is involved in Arabidopsis development, and its homologues from man and Mycobacterium leprae.     

Identification of higher plant GlsA,a putative morphogenesis factor of gametic cells

GlsA has been identified in an asexual-reproductive-cell (gonidia)-deficient mutant of Volvox as a chaperone-like protein essential for gonidia production. In this study, we isolated an angiosperm glsA (LlglsA) gene expressed during Lilium longiflorum pollen development. Immunoblot analyses showed that the strong LlGlsA expression occurred in the generative cell and its pattern during pollen development corresponded to that of alpha-tubulin. Morphological analyses succeeded in visualizing the dispersion of the strong LlGlsA signal in developing generative cells. In addition, multiple-immunofluorescence staining of LlGlsA and alpha-tubulin revealed that some of the dot-like LlGlsA signals were co-localized with microtubule filaments. From those results, we suggest that angiosperm GlsA functions as a chaperone modifying various structures during male gametic cell formation.     

Purification of a plant cell wall fibronectin-like adhesion protein involved in plant response to salt stress

The structural role of extracellular-matrix (ECM) has been recognized in both plants and animals as a support and anchorage-inducing cell behavior. Unlike the animal ECM proteins, the proteins that have been identified in plant ECM have not yet been purified from whole plants and cell wall. As several immunological data indicate the presence of animal ECM-like proteins in plants cell wall, especially under salt stress or water deficit, we propose a protocol to purify a fibronectin-like protein from the cell wall of epicotyls of young germinating peas. The process consists of a combination of gelatin and heparin affinity chromatography, close to the classical one used for human blood plasma fibronectin purification. Proteins with affinity for gelatin and heparin, immunologically related to human fibronectin, are found in the cell wall of epicotyls grown under salt stress or not. Total amount of purified proteins is 3-4 times more enriched in salt stressed epicotyls. SDS-PAGE and Western blot with antibodies directed against human blood plasma fibronectin give evidence that the cell wall proteins purified by gelatin/heparin affinity chromatography are closely related to human fibronectin. The present protocol leads us to purify 17 (control) or 65 (salt stress) micrograms of protein per g of fresh starting material. Our results suggest that plant cell wall proteins can provide better anchorage of the cell to its cell-wall during salt stress or water deficit and could be considered not only as cell adhesion but also as signaling molecules.     

Candida albicans Sun41p, a putative glycosidase, is involved in morphogenesis, cell wall biogenesis, and biofilm formation

The SUN gene family has been defined in Saccharomyces cerevisiae and comprises a fungus-specific family of proteins which show high similarity in their C-terminal domains. Genes of this family are involved in different cellular processes, like DNA replication, aging, mitochondrial biogenesis, and cytokinesis. In Candida albicans the SUN family comprises two genes, SUN41 and SIM1. We demonstrate that C. albicans mutants lacking SUN41 show similar defects as found for S. cerevisiae, including defects in cytokinesis. In addition, the SUN41 mutant showed a higher sensitivity towards the cell wall-disturbing agent Congo red, whereas no difference was observed in the presence of calcofluor white. Compared to the wild type, SUN41 deletion strains exhibited a defect in biofilm formation, a reduced adherence on a Caco-2 cell monolayer, and were unable to form hyphae on solid medium under the conditions tested. Interestingly, Sun41p was found to be secreted in the medium of cells growing as blastospores as well as those forming hyphae. Our results support a function of SUN41p as a glycosidase involved in cytokinesis, cell wall biogenesis, adhesion to host tissue, and biofilm formation, indicating an important role in the host-pathogen interaction.     

Umbraviruses--the unique plant viruses that do not encode a capsid protein

Umbraviruses are plant viruses that are unusual in that they lack within their genomes information for a capsid protein, and thereby for aphid transmission. They are transmitted by mechanical inoculation, but may become aphid-transmissible when the plants are co-infected with suitable luteoviruses which act as the helper viruses. In mixed infection the umbravirus can be encapsidated by the capsid protein shell provided from the luteovirus helper and, as a result, gain aphid transmissibility. The associations of some umbraviruses with luteoviruses result in specific, lasting disease complexes, showing interesting biological properties. However, umbraviruses are generally not sufficiently recognized, although several of them are significant pathogens in some regions of the world, including Europe. This review describes the development of studies upon umbraviruses and characterizes the genus Umbravirus and its best recognized members.     

The fixABCX genes in Rhodospirillum rubrum encode a putative membrane complex participating in electron transfer to nitrogenase

In our efforts to identify the components participating in electron transport to nitrogenase in Rhodospirillum rubrum, we used mini-Tn5 mutagenesis followed by metronidazole selection. One of the mutants isolated, SNT-1, exhibited a decreased growth rate and about 25% of the in vivo nitrogenase activity compared to the wild-type values. The in vitro nitrogenase activity was essentially wild type, indicating that the mutation affects electron transport to nitrogenase. Sequencing showed that the Tn5 insertion is located in a region with a high level of similarity to fixC, and extended sequencing revealed additional putative fix genes, in the order fixABCX. Complementation of SNT-1 with the whole fix gene cluster in trans restored wild-type nitrogenase activity and growth. Using Western blotting, we demonstrated that expression of fixA and fixB occurs only under conditions under which nitrogenase also is expressed. SNT-1 was further shown to produce larger amounts of both ribulose 1,5-bisphosphate carboxylase/oxygenase and polyhydroxy alkanoates than the wild type, indicating that the redox status is affected in this mutant. Using Western blotting, we found that FixA and FixB are soluble proteins, whereas FixC most likely is a transmembrane protein. We propose that the fixABCX genes encode a membrane protein complex that plays a central role in electron transfer to nitrogenase in R. rubrum. Furthermore, we suggest that FixC is the link between nitrogen fixation and the proton motive force generated in the photosynthetic reactions.     

Hyaluronan-binding protein in endothelial cell morphogenesis

Previous studies from several laboratories have provided evidence that interaction of hyaluronan (HA) with the surface of endothelial cells may be involved in endothelial cell behavior. We have recently characterized a mAb, mAb IVd4, that recognizes and neutralizes HA-binding protein (HABP) from a wide variety of cell types from several different species (Banerjee, S. D., and B. P. Toole. 1991. Dev. Biol. 146:186-197). In this study we have found that mAb IVd4 inhibits migration of endothelial cells from a confluent monolayer after "wounding" of the monolayer. HA hexasaccharide, a fragment of HA with the same disaccharide composition as polymeric HA, also inhibits migration. In addition, both reagents inhibit morphogenesis of capillary-like tubules formed in gels consisting of type I collagen and basement membrane components. Immunocytology revealed that the antigen recognized by mAb IVd4 becomes localized to the cell membrane of migrating cells, including many of their lamellipodia. Treatment with high concentrations of HA hexamer causes loss of immunoreactivity from these structures. We conclude that HABP recognized by mAb IVd4 is involved in endothelial cell migration and tubule formation.     

Crystal structure of the YjeE protein from Haemophilus influenzae: a putative Atpase involved in cell wall synthesis

A hypothetical protein encoded by the gene YjeE of Haemophilus influenzae was selected as part of a structural genomics project for X-ray analysis to assist with the functional assignment. The protein is considered essential to bacteria because the gene is present in virtually all bacterial genomes but not in those of archaea or eukaryotes. The amino acid sequence shows no homology to other proteins except for the presence of the Walker A motif G-X-X-X-X-G-K-T that indicates the possibility of a nucleotide-binding protein. The YjeE protein was cloned, expressed, and the crystal structure determined by the MAD method at 1.7-A resolution. The protein has a nucleotide-binding fold with a four-stranded parallel beta-sheet flanked by antiparallel beta-strands on each side. The topology of the beta-sheet is unique among P-loop proteins and has features of different families of enzymes. Crystallization of YjeE in the presence of ATP and Mg2+ resulted in the structure with ADP bound in the P-loop. The ATPase activity of YjeE was confirmed by kinetic measurements. The distribution of conserved residues suggests that the protein may work as a "molecular switch" triggered by ATP hydrolysis. The phylogenetic pattern of YjeE suggests its involvement in cell wall biosynthesis.     

CDC43 and RAM2 encode the polypeptide subunits of a yeast type I protein geranylgeranyltransferase.

The question regarding the identity of the alpha and beta subunits of the yeast type I protein geranylgeranyltransferase was explored using prokaryotic expression of candidate genes. The Saccharomyces cerevisiae CDC43 and RAM2 genes were expressed in Escherichia coli and cell extracts examined for the ability to transfer [3H]geranylgeranyl diphosphate to an appropriate CaaX protein substrate. Individual expression of each gene yielded no activity; however, co-expression of the two genes resulted in high levels of [3H] geranylgeranyl incorporation into the substrate protein Ras-Cys-Val-Val-Leu. The activity was partially purified yielding approximately 12,600 units/liter. The partially purified enzyme geranylgeranylated the Ras-Cys-Val-Val-Leu, Ras-Cys-Ala-Ile-Leu, Ras-Cys-Ile-Ile-Leu, and Ras-Cys-Thr-Ile-Leu substrates but not the Ras-Cys-Val-Leu-Ser or Ras-Ser-Val-Leu-Ser substrates. The protein geranylgeranyltransferase was highly specific for geranylgeranyl diphosphate and poorly transferred farnesyl. The recombinant enzyme was indistinguishable from the native type I geranylgeranyltransferase in yeast extracts. As has been reported for the protein farnesyltransferase, the yeast type I protein geranylgeranyltransferase is also a magnesium-requiring, zinc metalloenzyme. Interestingly, the recombinant enzyme functioned with calcium as the only divalent cation, although addition of zinc increased calcium-dependent activity 2-fold.     

Prion protein is a component of the multimolecular signaling complex involved in T cell activation

In this study we analyzed the interaction of prion protein PrP^C with components of glycosphingolipid-enriched microdomains in lymphoblastoid T cells. PrP^C was distributed in small clusters on the plasma membrane, as revealed by immunoelectron microscopy. PrP^C is present in microdomains, since it coimmunoprecipitates with GM3 and the raft marker GM1. A strict association between PrP^C and Fyn was revealed by scanning confocal microscopy and coimmunoprecipitation experiments. The phosphorylation protein ZAP-70 was immunoprecipitated by anti-PrP after T cell activation. These results demonstrate that PrP^C interacts with ZAP-70, suggesting that PrP^C is a component of the multimolecular signaling complex within microdomains involved in T cell activation.     

A small G protein Rhb1 and a GTPase-activating protein Tsc2 involved in nitrogen starvation-induced morphogenesis and cell wall integrity of Candida albicans

Rheb is a new member of the small G proteins of the Ras superfamily in eukaryotic organisms and controls various physiological processes. Activity of Rheb is regulated by Tsc2, a GTPase-activating protein (GAP). In this study, we have identified Candida albicans homologs of Rheb (named as Rhb1) and Tsc2. Deletion of the RHB1 gene showed enhanced sensitivity to rapamycin (an inhibitor of TOR kinase), suggesting that Rhb1 is associated with the TOR signaling pathway in C. albicans. Further analysis indicated RHB1 and TSC2 are involved in nitrogen starvation-induced filamentation, likely by controlling the expression of MEP2 whose gene product is an ammonium permease and a sensor for the nitrogen signal. Moreover, we have demonstrated that Rhb1 is also involved in cell wall integrity pathway, by transferring signals through the TOR kinase and the Mkc1 MAP kinase pathway. Together, this study brings new insights into the complex interplay of signaling and regulatory pathways in C. albicans.