A thiocyanate-forming protein generates multiple products upon allylglucosinolate breakdown in Thlaspi arvense

Glucosinolates, amino acid-derived thioglycosides found in plants of the Brassicales order, are one of the best studied classes of plant secondary metabolites. Together with myrosinases and supplementary proteins known as specifier proteins, they form the glucosinolate–myrosinase system that upon tissue damage gives rise to a number of biologically active glucosinolate breakdown products such as isothiocyanates, epithionitriles and organic thiocyanates involved in plant defense. While isothiocyanates are products of the spontaneous rearrangement of the glucosinolate aglycones released by myrosinase, the formation of epithionitriles and organic thiocyanates depends on both myrosinases and specifier proteins. Hydrolysis product profiles of many glucosinolate-containing plant species indicate the presence of specifier proteins, but only few have been identified and characterized biochemically. Here, we report on cDNA cloning, heterologous expression and characterization of TaTFP, a thiocyanate-forming protein (TFP) from Thlaspi arvense L. (Brassicaceae), that is expressed in all plant organs and can be purified in active form after heterologous expression in Escherichia coli. As a special feature, this protein promotes the formation of allylthiocyanate as well as the corresponding epithionitrile upon myrosinase-catalyzed hydrolysis of allylglucosinolate, the major glucosinolate of T. arvense. All other glucosinolates tested are converted to their simple nitriles when hydrolyzed in the presence of TaTFP. Despite its ability to promote allylthiocyanate formation, TaTFP has a higher amino acid sequence similarity to known epithiospecifier proteins (ESPs) than to Lepidium sativum TFP. However, unlike Arabidopsis thaliana ESP, its activity in vitro is not strictly dependent on Fe²⁺ addition to the assay mixtures. The availability of TaTFP in purified form enables future studies to be aimed at elucidating the structural bases of specifier protein specificities and mechanisms. Furthermore, identification of TaTFP shows that product specificities of specifier proteins can not be predicted based on amino acid sequence similarity and raises interesting questions about specifier protein evolution.     

The role of summer precipitation and summer temperature in establishment and growth of dwarf shrub <Emphasis Type="Italic">Betula nana</Emphasis> in northeast Siberian tundra

It is widely believed that deciduous tundra-shrub dominance is increasing in the pan-Arctic region, mainly due to rising temperature. We sampled dwarf birch (Betula nana L.) at a northeastern Siberian tundra site and used dendrochronological methods to explore the relationship between climatic variables and local shrub dominance. We found that establishment of shrub ramets was positively related to summer precipitation, which implies that the current high dominance of B. nana at our study site could be related to high summer precipitation in the period from 1960 to 1990. The results confirmed that early summer temperature is most influential to annual growth rates of B. nana. In addition, summer precipitation stimulated shrub growth in years with warm summers, suggesting that B. nana growth may be co-limited by summer moisture supply. The dual controlling role of temperature and summer precipitation on B. nana growth and establishment is important to predict future climate-driven vegetation dynamics in the Arctic tundra.     

Identification of Reggie-1 and Reggie-2 as plasmamembrane-associated proteins which cocluster with activated GPI-anchored cell adhesion molecules in non-caveolar micropatches in neurons

Neurons are believed to possess plasmalemmal microdomains and proteins analogous to the caveolae and caveolin of nonneuronal cells. Caveolae are plasmalemmal invaginations where activated glycosyl-phosphatidylinositol (GPI)-anchored proteins preferentially assemble and where transmembrane signaling may occur. Molecular cloning of rat reggie-1 and -2 (80% identical to goldfish reggie proteins) shows that reggie-2 is practically identical to mouse flotillin-1. Flotillin-1 and epidermal surface antigen (ESA) (flotillin-2) are suggested to represent possible membrane proteins in caveolae. Rat reggie-1 is 99% homologous to ESA in overlapping sequences but has a 49-amino-acid N-terminus not present in ESA. Antibodies (ABs) which recognize reggie-1 or -2 reveal that both proteins cluster at the plasmamembrane and occur in micropatches in neurons [dorsal root ganglia (DRGs), retinal ganglion, and PC-12 cells] and in nonneuronal cells. In neurons, reggie micropatches occur along the axon and in lamellipodia and filopodia of growth cones, but they do not occur in caveolae. By quantitative electronmicroscopic analysis we demonstrate the absence of caveolae in (anti-caveolin negative) neurons and show anti-reggie-1 immunogold-labeled clusters at the plasmamembrane of DRGs. When ABs against the GPI-anchored cell adhesion molecules (CAMs) F3 and Thy-1 are applied to live DRGs, the GPI-linked CAMs sequester into micropatches. Double immunofluorescence shows a colocalization of the CAMs with micropatches of anti-reggie antibodies. Thus, reggie-1 and reggie-2 identify sites where activated GPI-linked CAMs preferentially accumulate and which may represent noncaveolar micropatches (domains). © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 502–523, 1998     

Splicing factor SRp30c interaction with Y-box protein-1 confers nuclear YB-1 shuttling and alternative splice site selection

The multifunctional DNA- and RNA-associated Y-box protein 1 (YB-1) specifically binds to splicing recognition motifs and regulates alternative splice site selection. Here, we identify the arginine/serine-rich SRp30c protein as an interacting protein of YB-1 by performing a two-hybrid screen against a human mesangial cell cDNA library. Co-immunoprecipitation studies confirm a direct interaction of tagged proteins YB-1 and SRp30c in the absence of RNA via two independent protein domains of YB-1. A high affinity interaction is conferred through the N-terminal region. We show that the subcellular YB-1 localization is dependent on the cellular SRp30c content. In proliferating cells, YB-1 localizes to the cytoplasm, whereas FLAG-SRp30c protein is detected in the nucleus. After overexpression of YB-1 and FLAG-SRp30c, both proteins are co-localized in the nucleus, and this requires the N-terminal region of YB-1. Heat shock treatment of cells, a condition under which SRp30c accumulates in stress-induced Sam68 nuclear bodies, abrogates the co-localization and YB-1 shuttles back to the cytoplasm. Finally, the functional relevance of the YB-1/SRp30c interaction for in vivo splicing is demonstrated in the E1A minigene model system. Here, changes in splice site selection are detected, that is, overexpression of YB-1 is accompanied by preferential 5' splicing site selection and formation of the 12 S isoform.     

The SPA1-like proteins SPA3 and SPA4 repress photomorphogenesis in the light

Suppressor of phyA-105 (SPA1) is a phytochrome A-specific signaling intermediate that acts as a light-dependent repressor of photomorphogenesis in Arabidopsis seedlings. SPA1 is part of a small gene family comprising three genes: SPA1-related 2 (SPA2), SPA1-related 3 (SPA3), and SPA1-related 4 (SPA4). Here, we investigate the functions of SPA3 and SPA4, two very closely related genes coding for proteins with 74% identical amino acids. Seedlings with mutations in SPA3 or SPA4 exhibit enhanced photomorphogenesis in the light, but show no phenotype in darkness. While there are small differences between the effects of spa3 and spa4 mutations, it is apparent that SPA3 and SPA4 function to inhibit light responses in continuous far-red, red, and blue light. Phytochrome A is necessary for all aspects of the spa4 mutant phenotype, suggesting that SPA4, like SPA1, acts specifically in phytochrome A signaling. Enhanced photoresponsiveness of spa3 mutants is also fully dependent on phytochrome A in far-red and blue light, but not in red light. Hence, SPA3 function in red light may be dependent on other phytochromes in addition to phytochrome A. Using yeast two-hybrid and in vitro interaction assays, we further show that SPA3 as well as SPA4 can physically interact with the constitutive repressor of light signaling COP1. Deletion analyses suggest that SPA3 and SPA4, like SPA1, bind to the coiled-coil domain of COP1. Taken together, our results have identified two new loci coding for negative regulators that may be involved in fine tuning of light responses by interacting with COP1.     

Reductive Dehalogenation of Brominated Phenolic Compounds by Microorganisms Associated with the Marine Sponge Aplysina aerophoba

Marine sponges are natural sources of brominated organic compounds, including bromoindoles, bromophenols, and bromopyrroles, that may comprise up to 12% of the sponge dry weight. Aplysina aerophoba sponges harbor large numbers of bacteria that can amount to 40% of the biomass of the animal. We postulated that there might be mechanisms for microbially mediated degradation of these halogenated chemicals within the sponges. The capability of anaerobic microorganisms associated with the marine sponge to transform haloaromatic compounds was tested under different electron-accepting conditions (i.e., denitrifying, sulfidogenic, and methanogenic). We observed dehalogenation activity of sponge-associated microorganisms with various haloaromatics. 2-Bromo-, 3-bromo-, 4-bromo-, 2,6-dibromo-, and 2,4,6-tribromophenol, and 3,5-dibromo-4-hydroxybenzoate were reductively debrominated under methanogenic and sulfidogenic conditions with no activity observed in the presence of nitrate. Monochlorinated phenols were not transformed over a period of 1 year. Debromination of 2,4,6-tribromophenol, and 2,6-dibromophenol to 2-bromophenol was more rapid than the debromination of the monobrominated phenols. Ampicillin and chloramphenicol inhibited activity, suggesting that dehalogenation was mediated by bacteria. Characterization of the debrominating methanogenic consortia by using terminal restriction fragment length polymorphism (TRFLP) and denaturing gradient gel electrophoresis analysis indicated that different 16S ribosomal DNA (rDNA) phylotypes were enriched on the different halogenated substrates. Sponge-associated microorganisms enriched on organobromine compounds had distinct 16S rDNA TRFLP patterns and were most closely related to the δ subgroup of the proteobacteria. The presence of homologous reductive dehalogenase gene motifs in the sponge-associated microorganisms suggested that reductive dehalogenation might be coupled to dehalorespiration.     

Bread matters: a national initiative to profile the genetic diversity of Australian wheat

The large and complex genome of wheat makes genetic and genomic analysis in this important species both expensive and resource intensive. The application of next-generation sequencing technologies is particularly resource intensive, with at least 17?Gbp of sequence data required to obtain minimal (1×) coverage of the genome. A similar volume of data would represent almost 40× coverage of the rice genome. Progress can be made through the establishment of consortia to produce shared genomic resources. Australian wheat genome researchers, working with Bioplatforms Australia, have collaborated in a national initiative to establish a genetic diversity dataset representing Australian wheat germplasm based on whole genome next-generation sequencing data. Here, we describe the establishment and validation of this resource which can provide a model for broader international initiatives for the analysis of large and complex genomes.     

Behavior and Properties of Mature Lytic Granules at the Immunological Synapse of Human Cytotoxic T Lymphocytes

Killing of virally infected cells or tumor cells by cytotoxic T lymphocytes requires targeting of lytic granules to the junction between the CTL and its target. We used whole-cell patch clamp to measure the cell capacitance at fixed intracellular [Ca²⁺] to study fusion of lytic granules in human CTLs. Expression of a fluorescently labeled human granzyme B construct allowed identification of lytic granule fusion using total internal reflection fluorescence microscopy. In this way capacitance steps due to lytic granule fusion were identified. Our goal was to determine the size of fusing lytic granules and to describe their behavior at the plasma membrane. On average, 5.02 ± 3.09 (mean ± s.d.) lytic granules were released per CTL. The amplitude of lytic granule fusion events was ~ 3.3 fF consistent with a diameter of about 325 nm. Fusion latency was biphasic with time constants of 15.9 and 106 seconds. The dwell time of fusing lytic granules was exponentially distributed with a mean dwell time of 28.5 seconds. Fusion ended in spite of the continued presence of granules at the immune synapse. The mobility of fusing granules at the membrane was indistinguishable from that of lytic granules which failed to fuse. While dwelling at the plasma membrane lytic granules exhibit mobility consistent with docking interspersed with short periods of greater mobility. The failure of lytic granules to fuse when visible in TIRF at the membrane may indicate that a membrane-confined reaction is rate limiting.     

Circadian clock adjustment to plant iron status depends on chloroplast and phytochrome function

Plant chloroplasts are not only the main cellular location for storage of elemental iron (Fe), but also the main site for Fe, which is incorporated into chlorophyll, haem and the photosynthetic machinery. How plants measure internal Fe levels is unknown. We describe here a new Fe‐dependent response, a change in the period of the circadian clock. In Arabidopsis, the period lengthens when Fe becomes limiting, and gradually shortens as external Fe levels increase. Etiolated seedlings or light‐grown plants treated with plastid translation inhibitors do not respond to changes in Fe supply, pointing to developed chloroplasts as central hubs for circadian Fe sensing. Phytochrome‐deficient mutants maintain a short period even under Fe deficiency, stressing the role of early light signalling in coupling the clock to Fe responses. Further mutant and pharmacological analyses suggest that known players in plastid‐to‐nucleus signalling do not directly participate in Fe sensing. We propose that the sensor governing circadian Fe responses defines a new retrograde pathway that involves a plastid‐encoded protein that depends on phytochromes and the functional state of chloroplasts.     

Utilization of acidic amino acids and their amides by pseudomonads: role of periplasmic glutaminase-asparaginase

The acidic amino acids (Asp, Glu) and their amides (Asn, Gln) support rapid growth of a variety of Pseudomonas strains when provided as the sole source of carbon and nitrogen. All key enzymes of glutamate metabolism were detected in P. fluorescence, with glutaminase and asparaginase showing the highest specific activities. A periplasmic glutaminase/asparaginase activity (PGA) was found in all pseudomonads examined, including a number of root-colonizing biocontrol strains. The enzyme was purified and shown to be identical with the ansB gene product described previously. In addition to PGA, P. fluorescens contains a cytoplasmic asparaginase with marked specificity for Asn. PGA is strongly and specifically induced by its substrates (Asn, Gln) but also by the reaction products (Asp, Glu). In addition, PGA is subject to efficient carbon catabolite repression by glucose and by citrate cycle metabolites. A mutant of P. putida KT2440 with a disrupted ansB gene was unable to utilize Gln, whereas growth of the mutant on other amino acids was normal.