The extended arms of DNA-binding domains: a tale of tails

DNA-binding domains (DBDs) frequently have N- or C-terminal tails, rich in lysine and/or arginine and disordered in free solution, that bind the DNA separately from and in the opposite groove to the folded domain. Is their role simply to increase affinity for DNA or do they have a role in specificity, that is, sequence recognition? One approach to answering this question is to analyze the contribution of such tails to the overall energetics of binding. It turns out that, despite similarities of amino acid sequence, three distinct categories of DBD extension exist: (i) those that are purely electrostatic and lack specificity, (ii) those that are largely non-electrostatic with a high contribution to specificity and (iii) those of mixed character that show sequence preference. Because in all cases the tails also increase the affinity for target DNA, they represent a crucial component of the machinery for selective gene activation or repression.     

The presence of RNA in a double helix inhibits its interaction with histone protein

The binding of core histones (H2A, H2B, H3, H4) to a circular plasmid DNA and to a circular DNA-RNA hybrid molecule of similar size has been compared. Circular hybrid molecules were formed from single stranded fd DNA by synthesis of the complimentary strand with ribonucleotides using wheat germ RNA polymerase II. Upon reconstitution of plasmid DNA circles with histone, the sedimentation profiles of the DNA remained sharp by increased several fold in rate. Material from the peak fractions of these sedimentations appeared to be condensed circular loops of nucleosomes when examined by electron microscopy (EM), and the mass ratio of DNA to histone (at the histone concentrations which produced the fastest sedimentations) was typical of native chromatin. In contrast, the sedimentation behavior of DNA-RNA hybrid circles after addition of histone remained unchanged except for a minor fraction which exhibited a broad and faster sedimentation rate. Examination by EM revealed that most of the molecules appeared identical to protein free hybrid circles while the minor, faster sedimenting fraction appeared to be two or more circles bound together by protein aggregates. Finally, a linear molecule consisting of about 3000 base pairs of duplex DNA covalently joined on both ends to 1500 base pairs of RNA-DNA hybrid helix was constructed. Reconstitution of this molecule with core histone showed nucleosome formation only on the central DNA duplex region. Isopycnic banding of fixed hybrid-histone mixtures showed that little or no histone had bound to the bulk of the full hybrid molecules. We suggest that the presence of RNA in a nucleic acid duplex inhibits the condensation of the duplex into a nucleosomal structure by histone.     

The need for alternative plant species interaction models

对植物物种相互作用备选模型的需求近年来,经典的Lotka-Volterra模型在分析和解释植物物种间竞争性相互作用方面的局限性愈发凸显。目前已经明确的3个问题是：(1)缺乏频率依赖性,其对于物种的长期共存至关重要;(2)需要考虑影响个体表现的未测量(通常不可测量)的变量(例如,土壤养分或病原体的空间变化);以及(3)需要将测量误差与生物变异度分开。本文改进了经典的Lotka-Volterra竞争模型以期突破模型的局限。同时,我们将8个备选模型与丹麦草本植物群落中羊茅(Festuca ovina)和细弱剪股颖 (Agrostis capillaris) 3年以上的盖度数据拟合,应用贝叶斯建模框架来确定模型改进是否提高了模型的性能,并提高了其预测群落动态并因此检验假设的能力。研究结果表明,纳入频率依赖性和测量误差极大地改进了模型性能,但将可能的未测量变量考虑在内却未能改进其性能。我们的研究结果强调了在植物群落动态的定量研究中比较备选模型的重要性。只有考虑可能的备选模型,我们才能确定驱动群落构建和变化的因子,并提高我们预测植物群落行为的能力。     

The plant pathogen Ralstonia solanacearum needs aerotaxis for normal biofilm formation and interactions with its tomato host

Ralstonia solanacearum is a soilborne pathogen that causes bacterial wilt of diverse plant species. To locate and infect host plant roots R. solanacearum needs taxis, the ability to move toward more favorable conditions. However, the specific signals that attract this pathogen were unknown. One candidate is aerotaxis, or energy taxis, which guides bacteria toward optimal intracellular energy levels. The R. solanacearum genome encodes two putative aerotaxis transducers. Cloned R. solanacearum aer1 and aer2 genes restored aerotaxis to an Escherichia coli aer mutant, demonstrating that both genes encode heterologously functional aerotaxis transducers. Site-directed mutants lacking aer1, aer2, or both aer1 and aer2 were significantly less able to move up an oxygen gradient than the wild-type parent strain; in fact, the aerotaxis of the aer mutants was indistinguishable from that of a completely nonmotile strain. Tomato plants inoculated with either the aer2 or the aer1/aer2 mutant had slightly delayed wilt disease development. Furthermore, the aer1/aer2 double mutant was significantly impaired in the ability to rapidly localize on tomato roots compared to its wild-type parent. Unexpectedly, all nonaerotactic mutants formed thicker biofilms on abiotic surfaces than the wild type. These results indicate that energy taxis contributes significantly to the ability of R. solanacearum to locate and effectively interact with its host plants.     

Metapopulation structure of a seed-predator weevil and its host plant in arms race coevolution

Although the importance of gene flow in the geographic structuring of host-parasite interactions has been well discussed, little is known about how dispersal drives the spatial dynamics of other types of coevolutionary interactions in nature. We evaluated the roles of gene flow in the geographically structured processes of a predator-prey arms race involving a seed-predatory weevil with a long mouthpart and its host camellia plant with a thick fruit coat. Molecular genetic analyses showed that both weevil and camellia populations were structured at a spatial scale of several kilometers. Importantly, the spatial pattern of the migration of weevils, but not that of camellias, imposed significant effects on the geographic configuration of the levels of coevolutionary escalation. This result suggests that even if migration is limited in one species (camellia), local coevolution with the other species that migrates between neighboring localities (weevil) can reduce the interpopulation difference in the local adaptive optima of the former species. Thus, gene flow of a species potentially homogenizes the local biological environments provided by the species and thereby promotes the evolutionary convergence of its coevolving counterparts. Consequently, by focusing on coevolutionary interactions in natural communities, "indirect" effects of gene flow on the adaptive divergence of organisms could be identified.     

Phosphorylation of both nucleoplasmin domains is required for activation of its chromatin decondensation activity

Nucleoplasmin (NP) is a histone chaperone involved in nucleosome assembly, chromatin decondensation at fertilization, and apoptosis. To carry out these activities NP has to interact with different types of histones, an interaction that is regulated by phosphorylation. Here we have identified a number of phosphorylated residues by mass spectrometry and generated mutants in which these amino acids are replaced by Asp to mimic the effect of phosphorylation. Our results show that, among the eight phosphoryl groups experimentally detected, four are located at the flexible N terminus, and the rest are found at the tail domain, flanking the nuclear localization signal. Phosphorylation-mimicking mutations render a recombinant protein as active in chromatin decondensation as hyperphosphorylated NP isolated from Xenopus laevis eggs. Comparison of mutants in which the core and tail domains of the protein were independently or simultaneously "activated" indicates that activation or phosphorylation of both protein domains is required for NP to efficiently extract linker-type histones from chromatin.     

More insights into the CCN3/Connexin43 interaction complex and its role for signaling

Connexin43 (Cx43) forms gap junction channels but also serves as a signaling center by binding to proteins via its C‐terminus. We have previously demonstrated that transfection of Cx43 leads to significantly reduced proliferation of placental tumor cells through upregulating and binding of the growth regulator CCN3 (NOV) at the C‐terminus of Cx43. Here, we combined fluorescence resonance energy transfer (FRET), co‐immunoprecipitation and proliferation and expression assays to characterize the interaction complex of Cx43 and CCN3. FRET measurements confirmed the interaction of CCN3 with wild‐type Cx43 (amino acids 1‐382) and with mutants of Cx43 truncated at the C‐terminus resulting in Cx43 proteins of amino acids 1‐374, 1‐273, 1‐264, 1‐257 in 293T cells. These results matched the co‐immunoprecipitation data. Interestingly, although FRET revealed distinct efficiencies in interaction of Cx43 with CCN3 for all deletion constructs only wild‐type Cx43 and one deletion construct (1‐374) led to increased CCN3 expression. Only these interactions which were associated with increased CCN3 expression resulted in a reduced cell proliferation. Our study provides evidence that only defined binding properties between Cx43 and CCN3 leading to an upregulation of CCN3 are needed for signaling. Furthermore, the data obtained by FRET analysis allowed us to model the 3D structure of the C‐terminus of Cx43 interacting with CCN3. J. Cell. Biochem. 110: 129–140, 2010. © 2010 Wiley‐Liss, Inc.     

Adaptive divergence of scaling relationships mediates the arms race between a weevil and its host plant

Coevolution of exaggerated morphologies between insects and plants is a well-known but poorly understood phenomenon in evolutionary biology. In the antagonistic interaction between a seed-predatory insect, the camellia weevil (Curculio camelliae), and its host plant, Japanese camellia (Camellia japonica), we examined the evolutionary trajectory of an exaggerated offensive trait of the weevil (rostrum length) in terms of scaling relationship. Sampling throughout Japan revealed that the ratio of the rostrum length to overall body size was correlated with the ratio of the pericarp thickness to overall fruit size across the localities. We found a geographical interpopulation divergence in a parameter pertaining to the allometric equation of rostrum length (the coefficient a in y=axb, where y and x denote rostrum and body lengths, respectively), and the pattern of geographical differentiation in the allometric coefficient was closely correlated with the variation in the pericarp thickness of Japanese camellia. Our results provide a novel example of a geographically diverged scaling relationship in an insect morphology resulting from a coevolutionary arms race with its host plant.     

Seed dispersal effectiveness in a plant–lizard interaction and its consequences for plant regeneration after disperser loss

Mutualistic disruptions, such as those promoted by the loss of seed dispersers, can have negative effects on the plant regeneration of those species that strongly depend upon them. In order to adequately assess how plant communities are affected by such disruptions, we need to know the importance of the dispersal phase, both in its quantitative and qualitative components. We examined this in the narrow interaction between the shrub Daphne rodriguezii and its (only) disperser, the lizard Podarcis lilfordi. We quantified fruit removal and the effect of fruit/seed-size selection, seed treatment in the disperser’s guts and seed deposition patterns on seedling emergence and survival. In the only locality in which lizards persist, they removed most fruits and showed preference for larger ones in one of the two study years. Seed treatment in lizard’s guts had no effect on germination, although it tended to reduce the effect of seed size on germination (differences between large vs. small seeds in seed germination were higher for non-ingested seeds). Probability of seedling emergence, but not survival, was higher in the locality with lizards. Dispersed seeds under heterospecific shrubs showed higher seedling survival than those under conspecifics in all localities, especially the year with higher rainfall. Our findings support that the movement of seeds to nurse shrubs by lizards is the most important component of the seed dispersal process in the only remaining locality where both species coexist.     

The interaction between plant competition and disease

It is well documented that pathogens can affect the survival, reproduction, and growth of individual plants. Drawing together insights from diverse studies in ecology and agriculture, we evaluate the evidence for pathogens affecting competitive interactions between plants of both the same and different species. Our objective is to explore the potential ecological and evolutionary consequences of such interactions. First, we address how disease interacts with intraspecific competition and present a simple graphical model suggesting that diverse outcomes should be expected. We conclude that the presence of pathogens may have either large or minimal effects on population dynamics depending on many factors including the density-dependent compensatory ability of healthy plants and spatial patterns of infection. Second, we consider how disease can alter competitive abilities of genotypes, and thus may affect the genetic composition of populations. These genetic processes feed back on population dynamics given trade-offs between disease resistance and other fitness components. Third, we examine how the effect of disease on interspecific plant interactions may have potentially far-reaching effects on community composition. A host-specific pathogen, for example, may alter a competitive hierarchy that exists between host and non-host species. Generalist pathogens can also induce indirect competitive interactions between host species. We conclude by highlighting lacunae in our current understanding and suggest that future studies should (1) examine a broader taxonomic range of pathogens since work to date has largely focused on fungal pathogens; (2) increase the use of field competition studies; (3) follow interactions for multiple generations; (4) characterize density-dependent processes; and (5) quantify pathogen, as well as plant, population and community dynamics.     

Improvement of plastic-based disposable bioreactors for plant science needs

The present article describes two new applications of plastic-based cell culture systems in the plant biotechnology domain. Different types of bioreactors are used at Nestlé R&D Center-Tours for large scale culture of plants cells to produce metabolites or recombinant proteins and for mass propagation of selected plant varieties by somatic embryogenesis. Particularly, recent studies are directed to cut down the production costs of these two processes by developing disposable cell culture systems. For large scale culture, two novel flexible plastic-based disposable bioreactors have been developed from 10 to 100 l working volumes, validated with several plant species (“Wave and Undertow” and “Slug Bubble” bioreactors). Vegetative propagation of elite plant varieties is achieved through somatic embryogenesis in liquid medium. A pilot scale process has been recently set up for the industrial propagation of Coffea canephora (Robusta coffee). The current production capacity is 2.5–3.0 million embryos per year. The pre-germination of the embryos was previously conducted by temporary immersion in liquid medium in 10-l glass bioreactors. An improved process has been developed using a 10-l disposable bioreactor consisting in a bag containing a rigid plastic box (“Box-in-Bag” bioreactor), insuring, amongst other advantages, a higher light transmittance to the biomass due to its horizontal design.     

The sodium pump needs its beta subunit

The sodium pump Na,K-ATPase, located in the plasma membrane of all animal cells, is a member of a family of ion-translocating ATPases that share highly homologous catalytic subunits. In this family, only Na,K-ATPase has been established to be a heterodimer of catalytic (alpha) and glycoprotein (beta) subunits. The beta subunit has not been associated with the pump's transport or enzymatic activity, and its role in Na,K-ATPase function has been, until recently, a puzzle. In this review we describe what is known about the structure of beta and summarize evidence that expression of both alpha and beta subunits is required for Na,K-ATPase activity, that inhibition of glycosylation causes a decrease in accumulation of both alpha and beta subunits, and we provide evidence that pretranslational up-regulation of beta alone can lead to increased abundance of sodium pumps. These findings are all consistent with the hypothesis that the beta subunit regulates, through assembly of alpha beta heterodimers, the number of sodium pumps transported to the plasma membrane.     

The proteasome regulates the interaction between Cx43 and ZO-1

Gap junction (GJ) intercellular communication (GJIC) is vital to ensure proper cell and tissue function. GJ are multimeric structures composed of proteins called connexins. Modifications on stability or subcellular distribution of connexins have a direct impact on the extent of GJIC. In this study we have investigated the role of the proteasome in regulation of connexin 43 (Cx43) internalization. Although the participation of both the proteasome and lysosome has long been suggested in Cx43 degradation, the molecular mechanisms whereby proteasome contributes to regulate Cx43 internalization and intercellular communication are still unclear. The results presented in this study envision a new mechanism whereby proteasome regulates GJIC by modulating interaction between Cx43 and ZO-1. Immunoprecipitation experiments, in the presence of proteasome inhibitors, together with immunofluorescence data indicate that the proteasome regulates interaction between Cx43 and ZO-1. Overexpression of the PDZ2 domain of ZO-1 and the expression of Cx-43 fused in frame with a V5/HIS tag, suggest that interaction between the two proteins occurs through the PDZ2 domain of ZO-1 and the C-terminus of Cx43. When interaction between Cx43 and ZO-1 is reduced, as in the presence of proteasome inhibitors, Cx43 accumulates, forming large GJ plaques at plasma membrane. Data presented in this article suggest a new pathway whereby alterations in proteasome activity may impact on GJIC as well as on non-junctional communication with extracellular environment, contributing to cell and tissue dysfunction.     

Phyllosphere microbiota: Community dynamics and its interaction with plant hosts

Plants are colonized by various microorganisms in natural environments. While many studies have demonstrated key roles of the rhizosphere microbiota in regulating biological processes such as nutrient acquisition and resistance against abiotic and biotic challenges, less is known about the role of the phyllosphere microbiota and how it is established and maintained. This review provides an update on current understanding of phyllosphere community assembly and the mechanisms by which plants and microbes establish the phyllosphere microbiota for plant health.     

Accessibility of histone H1° and its structural domains to antibody binding in extended and folded chromatin

The aim of this work was to study the accessibility of histone H1° and its structural domains to antibody binding in high molecular mass chromatin fragments of different conformations. Three types of specific antibody populations were used: (1) anti-H1° which reacted with antigenic determinants situated along the whole polypeptide chain, (2) anti-GH5 or anti-GH1° which recognized epitopes located in the globular region of H1° and (3) anti-C-tail antibodies reacting specifically with fragment 99–193 of the protein molecule. The immunoreactivity of the chromatin-bound antigen was investigated by solid-phase ELISA performed on glutaraldehyde-cross-linked chromatin and by an inhibition assay carried out with native chromatin in solution. The results of both methods were unidirectional and showed that: (1) the accessibility of H1° did not change with the compaction of the fiber; (2) the G-domain was not accessible to antibodies either in the relaxed or in the condensed state of the fragments, (3) the binding of the C-terminus-specific antibodies was different for isolated monosomes and for the chromatin fiber and (4) the degree of exposure of the epitopes of H1° in chromatin was much less than that of histone H1.Abbreviations ELISA Enzyme-Linked Immunosorbent Assay - G-domain Globular domain - IgG Immunoglobulin G - SDS Sodium Dodecylsulphate