Identification of immunologically related proteins in sieve-tube exudate collected from monocotyledonous and dicotyledonous plants

The mature, functional sieve-tube system in higher plants is dependent upon protein import from the companion cells to maintain a functional long-distance transport system. Soluble proteins present within the sieve-tube lumen were investigated by analysis of sieve-tube exudates which revealed the presence of distinct sets of polypeptides in seven monocotyledonous and dicotyledonous plant species. Antibodies directed against sieve-tube exudate proteins from Ricinus communis L. demonstrated the presence of shared antigens in the phloem sap collected from Triticum aestivum L., Oryza sativa L., Yucca filamentosa L., Cucurbita maxima Duch., Robinia pseudoacacia L. and Tilia platyphyllos L. Specific antibodies were employed to identify major polypeptides. Molecular chaperones related to Rubisco-subunit-binding protein and cyclophilin, as well as ubiquitin and the redox proteins, thioredoxin h and glutaredoxin, were detected in the sieve-tube exudate of all species examined. Actin and profilin, a modulator of actin polymerization, were also present in all analyzed phloem exudates. However, some proteins were highly species-specific, e.g. cystatin, a protease-inhibitor was present in R. communis but was not detected in exudates from other species, and orthologs of the well-known squash phloem lectin, phloem protein 2, were only identified in the sieve-tube exudate of R. communis and R. pseudoacacia. These findings are discussed in terms of the likely roles played by phloem proteins in the maintenance and function of the enucleate sieve-tube system of higher plants. Received: 12 February 1998 / Accepted: 16 March 1998     

Construction and characterisation of a gridded chicken cosmid library with four-fold genomic coverage

Gridded genomic libraries are crucial for the positional candidate gene approach. For this purpose we constructed a gridded genomic library from a female chicken using the vector sCos 1. About 110 000 cosmid clones were grown and replicated in 384-well plates. An average insert size of 39 kb was calculated from the analysis of 68 randomly selected clones. No chimerism could be observed from 31 in situ hybridisations. One replica of the library (number 125) has been transferred to the Resource Centre/Primary Database (RZPD) of the German Human Genome Project (DHGP). The whole library was gridded onto four nylon filters at high density for efficient identification of cosmid clones by colony hybridisation. Twenty-two probes were used for screening the library and each of them gave at least one positive signal. This result is in good agreement with a four-fold coverage of the genome as estimated from the insert length and number of recombinant clones. This library provides a powerful tool for rapid physical mapping and complex analysis of the chicken genome.     

Peptide labeling with photoactivatable trifunctional cadaverine derivative and identification of interacting partners by biotin transfer

A new photoactivatable trifunctional cross-linker, cBED (cadaverine-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3′-dithiopropionate), was synthesized by chemical conversion of sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3′-dithiopropionate) with cadaverine. This cross-linker was purified by reversed-phase high-performance liquid chromatography (RP–HPLC) and characterized using matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) analysis. cBED is based on sulfo-SBED that has a photoactivatable azido group, a cleavable disulfide bond for label transfer methods, and a biotin moiety for highly sensitive biotin/avidin detection. By ultraviolet (UV) light, the azido group is converted to a reactive nitrene, transforming transient bindings of interacting structures to covalent bonds. In contrast to the sulfo-N-hydroxysuccinimide (sulfo-NHS) moiety of sulfo-SBED, which attaches quite unspecifically to amino groups, cBED includes a cadaverine moiety that can be attached by transglutaminase more specifically to certain glutamine residues. For instance, thymosin β₄ can be labeled with cBED using tissue transglutaminase. By high-resolution HPLC/ESI–MS (electrospray ionization–mass spectrometry) and tandem MS (MS/MS) of the trypsin digest, it was established that glutamine residues at positions 23 and 36 were labeled, whereas Q39 showed no reactivity. The covalent binding of cBED to thymosin β₄ did not influence its G-actin sequestering activity, and the complex could be used to identify new interaction partners. Therefore, cBED can be used to better understand the multifunctional role of thymosin β₄ as well as of other proteins and peptides.     

Evolutionary biology and the treatment of signs and symptoms of infectious disease

When viewed from an evolutionary perspective, manifestations of infectious diseases can be classified as (1) adaptations of the host to counteract harmful aspects of the disease, (2) adaptations of the pathogen to manipulate the host, or (3) “side effects” of the disease that do not serve adaptive functions for either the host or the pathogen. Although the functions of most manifestations are not known, support or rejection of these hypotheses should be readily derivable in many cases from analyses of existing data and relatively simple experiments. This approach should lead to improved medical treatment because preferred treatment depends on assessment of the validity of the three explanations. As an illustration, this perspective and its consequences for therapy are analyzed for fever, rhinorrhea and diarrhea.     

The anatomy of the stigma and style from Cyclamen persicum (Mill.) cv. "pure white" and its relation to pollination success

Old flowers of Cyclamen generate few or no seeds. To understand the pollination problems of Cyclamen we investigate the general anatomy of the stigma and the style of Cyclamen persicum by scanning electron microscopy at different stages of floral maturity. Our investigations confirm that there is a hollow style. Against data commonly found in the literature, we present evidence of pollen germination and tube growth that show the stigma is not outside the style but inside it. Furthermore the maturation process of the style during the flowering time indicates a mechanism by which the stigma becomes shut off through closure at the terminal aperture of the style. At 3 to 5 days after anthesis there was the beginning closure of the style which was nearly completed at 21 days. The substance which leads to the closure is still unknown. The closure of the hollow style is a probable cause for failure of seed set in flowers not pollinated early in anthesis.     

Global trade will accelerate plant invasions in emerging economies under climate change

Trade plays a key role in the spread of alien species and has arguably contributed to the recent enormous acceleration of biological invasions, thus homogenizing biotas worldwide. Combining data on 60‐year trends of bilateral trade, as well as on biodiversity and climate, we modeled the global spread of plant species among 147 countries. The model results were compared with a recently compiled unique global data set on numbers of naturalized alien vascular plant species representing the most comprehensive collection of naturalized plant distributions currently available. The model identifies major source regions, introduction routes, and hot spots of plant invasions that agree well with observed naturalized plant numbers. In contrast to common knowledge, we show that the ‘imperialist dogma,’ stating that Europe has been a net exporter of naturalized plants since colonial times, does not hold for the past 60 years, when more naturalized plants were being imported to than exported from Europe. Our results highlight that the current distribution of naturalized plants is best predicted by socioeconomic activities 20 years ago. We took advantage of the observed time lag and used trade developments until recent times to predict naturalized plant trajectories for the next two decades. This shows that particularly strong increases in naturalized plant numbers are expected in the next 20 years for emerging economies in megadiverse regions. The interaction with predicted future climate change will increase invasions in northern temperate countries and reduce them in tropical and (sub)tropical regions, yet not by enough to cancel out the trade‐related increase.     

Ecological preferences of alien plant species in North-Eastern Germany

The large, comprehensive vegetation database of Mecklenburg-Vorpommern/NE Germany with 51,328 relevés allowed us to study an entire regional flora of 133 non-native plants (NNP, immigration after 1492 AD) with regard to their preferences to all kinds of habitats and along different ecological gradients. For each relevé, we computed average Ellenberg indicator values (EIV) for temperature, light, moisture, reaction, nutrients and salt as well as plant strategy type weights. We partitioned the dataset into relevés with and without occurrences of NNP and compared them with respect to the relative frequencies of EIVs and strategy type weights. We identified deviations from random differences by testing against permuted indicator values. To account for bias in EIV between community types, NNP preferences were differentiated for 34 phytosociological classes. We tested significance of preferences for the group of NNP as a whole, as well as for single NNP species within the entire dataset, as well as differentiated by phytosociological classes and formations. NNP as a group prefer communities with high EIVs for temperature and nutrients and low EIVs for moisture. They avoid communities with low EIV for reaction and high EIV for salt. NNP prefer communities with high proportions of ruderal and low proportion of stress strategists. The differentiation by phytosociological classes reinforces the general trends for temperature, nutrients, moisture, R and S strategy types. Nevertheless, preferences of single species reveal that NNP are not a congruent group but show individualistic ecological preferences.     

Engineering genetically encoded nanosensors for real-time in vivo measurements of citrate concentrations

Citrate is an intermediate in catabolic as well as biosynthetic pathways and is an important regulatory molecule in the control of glycolysis and lipid metabolism. Mass spectrometric and NMR based metabolomics allow measuring citrate concentrations, but only with limited spatial and temporal resolution. Methods are so far lacking to monitor citrate levels in real-time in-vivo. Here, we present a series of genetically encoded citrate sensors based on Förster resonance energy transfer (FRET). We screened databases for citrate-binding proteins and tested three candidates in vitro. The citrate binding domain of the Klebsiella pneumoniae histidine sensor kinase CitA, inserted between the FRET pair Venus/CFP, yielded a sensor highly specific for citrate. We optimized the peptide linkers to achieve maximal FRET change upon citrate binding. By modifying residues in the citrate binding pocket, we were able to construct seven sensors with different affinities spanning a concentration range of three orders of magnitude without losing specificity. In a first in vivo application we show that E. coli maintains the capacity to take up glucose or acetate within seconds even after long-term starvation.