Ubiquitin-dependent proteolytic control of SUMO conjugates

Posttranslational protein modification with small ubiquitin-related modifier (SUMO) is an important regulatory mechanism implicated in many cellular processes, including several of biomedical relevance. We report that inhibition of the proteasome leads to accumulation of proteins that are simultaneously conjugated to both SUMO and ubiquitin in yeast and in human cells. A similar accumulation of such conjugates was detected in Saccharomyces cerevisiae ubc4 ubc5 cells as well as in mutants lacking two RING finger proteins, Ris1 and Hex3/Slx5-Slx8, that bind to SUMO as well as to the ubiquitin-conjugating enzyme Ubc4. In vitro, Hex3-Slx8 complexes promote Ubc4-dependent ubiquitylation. Together these data identify a previously unrecognized pathway that mediates the proteolytic down-regulation of sumoylated proteins. Formation of substrate-linked SUMO chains promotes targeting of SUMO-modified substrates for ubiquitin-mediated proteolysis. Genetic and biochemical evidence indicates that SUMO conjugation can ultimately lead to inactivation of sumoylated substrates by polysumoylation and/or ubiquitin-dependent degradation. Simultaneous inhibition of both mechanisms leads to severe phenotypic defects.     

<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> is a susceptible host plant for the holoparasite <Emphasis Type="Italic">Cuscuta </Emphasis>spec

Arabidopsis thaliana and Cuscuta spec. represent a compatible host–parasite combination. Cuscuta produces a haustorium that penetrates the host tissue. In early stages of development the searching hyphae on the tip of the haustorial cone are connected to the host tissue by interspecific plasmodesmata. Ten days after infection, translocation of the fluorescent dyes, Texas Red (TR) and 5,6-carboxyfluorescein (CF), demonstrates the existence of a continuous connection between xylem and phloem of the host and parasite. Cuscuta becomes the dominant sink in this host–parasite system. Transgenic Arabidopsis plants expressing genes encoding the green fluorescent protein (GFP; 27 kDa) or a GFP–ubiquitin fusion (36 kDa), respectively, under the companion cell (CC)-specific AtSUC2 promoter were used to monitor the transfer of these proteins from the host sieve elements to those of Cuscuta. Although GFP is transferred unimpedly to the parasite, the GFP–ubiquitin fusion could not be detected in Cuscuta. A translocation of the GFP–ubiquitin fusion protein was found to be restricted to the phloem of the host, although a functional symplastic pathway exists between the host and parasite, as demonstrated by the transport of CF. These results indicate a peripheral size exclusion limit (SEL) between 27 and 36 kDa for the symplastic connections between host and Cuscuta sieve elements. Forty-six accessions of A. thaliana covering the entire range of its genetic diversity, as well as Arabidopsis halleri, were found to be susceptible towards Cuscuta reflexa.     

Profiling of Arabidopsis secondary metabolites by capillary liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry

Large-scale metabolic profiling is expected to develop into an integral part of functional genomics and systems biology. The metabolome of a cell or an organism is chemically highly complex. Therefore, comprehensive biochemical phenotyping requires a multitude of analytical techniques. Here, we describe a profiling approach that combines separation by capillary liquid chromatography with the high resolution, high sensitivity, and high mass accuracy of quadrupole time-of-flight mass spectrometry. About 2000 different mass signals can be detected in extracts of Arabidopsis roots and leaves. Many of these originate from Arabidopsis secondary metabolites. Detection based on retention times and exact masses is robust and reproducible. The dynamic range is sufficient for the quantification of metabolites. Assessment of the reproducibility of the analysis showed that biological variability exceeds technical variability. Tools were optimized or established for the automatic data deconvolution and data processing. Subtle differences between samples can be detected as tested with the chalcone synthase deficient tt4 mutant. The accuracy of time-of-flight mass analysis allows to calculate elemental compositions and to tentatively identify metabolites. In-source fragmentation and tandem mass spectrometry can be used to gain structural information. This approach has the potential to significantly contribute to establishing the metabolome of Arabidopsis and other model systems. The principles of separation and mass analysis of this technique, together with its sensitivity and resolving power, greatly expand the range of metabolic profiling.     

Dynamic changes in the localization of MAPK cascade components controlling pathogenesis-related (PR) gene expression during innate immunity in parsley

The activation of mitogen-activated protein kinase (MAPK) cascades is an important mechanism for stress adaptation through the control of gene expression in mammals, yeast, and plants. MAPK activation has emerged as a common mechanism by which plants trigger pathogen defense responses following innate immune recognition of potential microbial pathogens. We are studying the non-host plant defense response of parsley to attempted infection by Phytophthora species using an experimental system of cultured parsley cells and the Phytophthora-derived Pep-13 peptide elicitor. Following receptor-mediated recognition of this peptide, parsley cells trigger a multifaceted innate immune response, involving the activation of three MAPKs that have been shown to function in the oxidative burst-independent activation of defense gene expression. Using this same experimental model we now report the identification of a MAPK kinase (MAPKK) that functions upstream in this pathway. This kinase, referred to as PcMKK5 based on sequence similarity to Arabidopsis thaliana AtMKK5, is activated in parsley cells following Pep-13 treatment and functions as an in vivo activator of all three MAPKs previously shown to be involved in this response. Gain- and loss-of-function mutant versions of PcMKK5, when used in protoplast co-transfection assays, demonstrated that kinase activity of PcMKK5 is required for PR gene promoter activation following Pep-13 treatment. Furthermore, using specific antibodies and immunofluorescent labeling, we demonstrate that activation of MAPKs in parsley cells correlates with an increase in their nuclear localization, which is not detectable for activated PcMKK5. These results suggest that activation of gene expression through MAPK cascades during innate immune responses in plants involves dynamic changes in the localization of the proteins involved, which may reflect the distribution of key protein substrates for the activated MAPKs.     

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Facies arrangement and cyclostratigraphic architecture of a shallow-marine,warm-water carbonate platform: the Late Carboniferous Ny Friesland Platform in eastern Spitsbergen (Pyefjellet Beds,Wordiekammen Formation,Gipsdalen Group)

During the Late Carboniferous, a spacious warm-water carbonate platform developed across the eastern part of the present Arctic archipelago of Svalbard. The platform initiated in the Moscovian on an uplifted fault block (Ny Friesland High) and progradated during the Late Moscovian to Early Kasimovian into the adjacent Campbellryggen Basin (central Spitsbergen). The fossiliferous platform strata are characterized by a pronounced cyclicity formed by stacked parasequences, which consist of defined, subtidal to supratidal facies-set successions reflecting a general shallowing of the depositional area. Up to 17 of these shallowing-upward cycles, bounded by distinct discontinuity (marine flooding) surfaces due to the recurrent emersion and subsequent flooding of the platform surface, have been recognized within the platform strata. The stacked cycles are the result of global, glacio-eustatic, high-frequent and high-amplitudinal sea-level fluctuations with eccentricity periodicities caused by ice volume changes during the Gondwana Land glaciation. Based on systematic changes of the cycles (thickness and internal facies composition), the upper part of the platform strata is interpreted as a progradational parasequence set of a late highstand system tract. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Response properties of motion-sensitive visual interneurons in the lobula plate of Drosophila melanogaster

The crystalline-like structure of the optic lobes of the fruit fly Drosophila melanogaster has made them a model system for the study of neuronal cell-fate determination, axonal path finding, and target selection. For functional studies, however, the small size of the constituting visual interneurons has so far presented a formidable barrier. We have overcome this problem by establishing in vivo whole-cell recordings from genetically targeted visual interneurons of Drosophila. Here, we describe the response properties of six motion-sensitive large-field neurons in the lobula plate that form a network consisting of individually identifiable, directionally selective cells most sensitive to vertical image motion (VS cells). Individual VS cell responses to visual motion stimuli exhibit all the characteristics that are indicative of presynaptic input from elementary motion detectors of the correlation type. Different VS cells possess distinct receptive fields that are arranged sequentially along the eye's azimuth, corresponding to their characteristic cellular morphology and position within the retinotopically organized lobula plate. In addition, lateral connections between individual VS cells cause strongly overlapping receptive fields that are wider than expected from their dendritic input. Our results suggest that motion vision in different dipteran fly species is accomplished in similar circuitries and according to common algorithmic rules. The underlying neural mechanisms of population coding within the VS cell network and of elementary motion detection, respectively, can now be analyzed by the combination of electrophysiology and genetic intervention in Drosophila.