Comprehensive analysis of CLE polypeptide signaling gene expression and overexpression activity in Arabidopsis

Intercellular signaling is essential for the coordination of growth and development in higher plants. Although hundreds of putative receptors have been identified in Arabidopsis (Arabidopsis thaliana), only a few families of extracellular signaling molecules have been discovered, and their biological roles are largely unknown. To expand our insight into the developmental processes potentially regulated by ligand-mediated signal transduction pathways, we undertook a systematic expression analysis of the members of the Arabidopsis CLAVATA3/ESR-RELATED (CLE) small signaling polypeptide family. Using reporter constructs, we show that the CLE genes have distinct and specific patterns of promoter activity. We find that each Arabidopsis tissue expresses at least one CLE gene, indicating that CLE-mediated signaling pathways are likely to play roles in many biological processes during the plant life cycle. Some CLE genes that are closely related in sequence have dissimilar expression profiles, yet in many tissues multiple CLE genes have overlapping patterns of promoter-driven reporter activity. This observation, plus the general absence of detectable morphological phenotypes in cle null mutants, suggest that a high degree of functional redundancy exists among CLE gene family members. Our work establishes a community resource of CLE-related biological materials and provides a platform for understanding and ultimately manipulating many different plant signaling systems.     

CLE14/CLE20 peptides may interact with CLAVATA2/CORYNE receptor-like kinases to irreversibly inhibit cell division in the root meristem of Arabidopsis

Towards an understanding of the interacting nature of the CLAVATA (CLV) complex, we predicted the 3D structures of CLV3/ESR-related (CLE) peptides and the ectodomain of their potential receptor proteins/kinases, and docking models of these molecules. The results show that the ectodomain of CLV1 can form homodimers and that the 12-/13-amino-acid CLV3 peptide fits into the binding clefts of the CLV1 dimers. Our results also demonstrate that the receptor domain of CORYNE (CRN), a recently identified receptor-like kinase, binds tightly to the ectodomain of CLV2, and this likely leads to an increased possibility for docking with CLV1. Furthermore, our docking models reveal that two CRN-CLV2 ectodomain heterodimers are able to form a tetramer receptor complex. Peptides of CLV3, CLE14, CLE19, and CLE20 are also able to bind a potential CLV2-CRN heterodimer or heterotetramer complex. Using a cell-division reporter line, we found that synthetic 12-amino-acid CLE14 and CLE20 peptides inhibit, irreversibly, root growth by reducing cell division rates in the root apical meristem, resulting in a short-root phenotype. Intriguingly, we observed that exogenous application of cytokinin can partially rescue the short-root phenotype induced by over-expression of either CLE14 or CLE20 in planta. However, cytokinin treatment does not rescue the short-root phenotype caused by exogenous application of the synthetic CLE14/CLE20 peptides, suggesting a requirement for a condition provided only in living plants. These results therefore imply that the CLE14/CLE20 peptides may act through the CLV2-CRN receptor kinase, and that their availabilities and/or abundances may be affected by cytokinin activity in planta.     

Endogenous H2O2 produced by Streptococcus pneumoniae controls FabF activity

FabF elongation condensing enzyme is a critical factor in determining the spectrum of products produced by the FASII pathway. Its active site contains a critical cysteine-thiol residue, which is a plausible target for oxidation by H₂O₂. Streptococcus pneumoniae produces exceptionally high levels of H₂O₂, mainly through the conversion of pyruvate to acetyl-P via pyruvate oxidase (SpxB). We present evidence showing that endogenous H₂O₂ inhibits FabF activity by specifically oxidizing its active site cysteine-thiol residue. Thiol trapping methods revealed that one of the three FabF cysteines in the wild-type strain was oxidized, whereas in an spxB mutant, defective in H₂O₂ production, none of the cysteines was oxidized, indicating that the difference in FabF redox state originated from endogenous H₂O₂. In vitro exposure of the spxB mutant to various H₂O₂ concentrations further confirmed that only one cysteine residue was susceptible to oxidation. By blocking FabF active site cysteine with cerulenin we show that the oxidized cysteine was the catalytic one. Inhibition of FabF activity by either H₂O₂ or cerulenin resulted in altered membrane fatty acid composition. We conclude that FabF activity is inhibited by H₂O₂ produced by S. pneumoniae.     

Ceramide synthases 2, 5, and 6 confer distinct roles in radiation-induced apoptosis in HeLa cells

The role of ceramide neo-genesis in cellular stress response signaling is gaining increasing attention with recent progress in elucidating the novel roles and biochemical properties of the ceramide synthase (CerS) enzymes. Selective tissue and subcellular distribution of the six mammalian CerS isoforms, combined with distinct fatty acyl chain length substrate preferences, implicate differential functions of specific ceramide species in cellular signaling. We report here that ionizing radiation (IR) induces de novo synthesis of ceramide to influence HeLa cell apoptosis by specifically activating CerS isoforms 2, 5, and 6 that generate opposing anti- and pro-apoptotic ceramides in mitochondrial membranes. Overexpression of CerS2 resulted in partial protection from IR-induced apoptosis whereas overexpression of CerS5 increased apoptosis in HeLa cells. Knockdown studies determined that CerS2 is responsible for all observable IR-induced C_24:0 CerS activity, and while CerS5 and CerS6 each confer ～ 50% of the C_16:0 CerS baseline synthetic activity, both are required for IR-induced activity. Additionally, co-immunoprecipitation studies suggest that CerS2, 5, and 6 might exist as heterocomplexes in HeLa cells, providing further insight into the regulation of CerS proteins. These data add to the growing body of evidence demonstrating interplay among the CerS proteins in a stress stimulus-, cell type- and subcellular compartment-specific manner.     

Genetic architecture of a feeding adaptation: garter snake (Thamnophis) resistance to tetrodotoxin bearing prey

Detailing the genetic basis of adaptive variation in natural populations is a first step towards understanding the process of adaptive evolution, yet few ecologically relevant traits have been characterized at the genetic level in wild populations. Traits that mediate coevolutionary interactions between species are ideal for studying adaptation because of the intensity of selection and the well-characterized ecological context. We have previously described the ecological context, evolutionary history and partial genetic basis of tetrodotoxin (TTX) resistance in garter snakes (Thamnophis). Derived mutations in a voltage-gated sodium channel gene (Na_v1.4) in three garter snake species are associated with resistance to TTX, the lethal neurotoxin found in their newt prey (Taricha). Here we evaluate the contribution of Na_v1.4 alleles to TTX resistance in two of those species from central coastal California. We measured the phenotypes (TTX resistance) and genotypes (Na_v1.4 and microsatellites) in a local sample of Thamnophis atratus and Thamnophis sirtalis. Allelic variation in Na_v1.4 explains 23 per cent of the variation in TTX resistance in T. atratus while variation in a haphazard sample of the genome (neutral microsatellite markers) shows no association with the phenotype. Similarly, allelic variation in Na_v1.4 correlates almost perfectly with TTX resistance in T. sirtalis, but neutral variation does not. These strong correlations suggest that Na_v1.4 is a major effect locus. The simple genetic architecture of TTX resistance in garter snakes may significantly impact the dynamics of phenotypic coevolution. Fixation of a few alleles of major effect in some garter snake populations may have led to the evolution of extreme phenotypes and an ‘escape’ from the arms race with newts.     

An Overview of Marine Biodiversity in United States Waters

Marine biodiversity of the United States (U.S.) is extensively documented, but data assembled by the United States National Committee for the Census of Marine Life demonstrate that even the most complete taxonomic inventories are based on records scattered in space and time. The best-known taxa are those of commercial importance. Body size is directly correlated with knowledge of a species, and knowledge also diminishes with distance from shore and depth. Measures of biodiversity other than species diversity, such as ecosystem and genetic diversity, are poorly documented. Threats to marine biodiversity in the U.S. are the same as those for most of the world: overexploitation of living resources; reduced water quality; coastal development; shipping; invasive species; rising temperature and concentrations of carbon dioxide in the surface ocean, and other changes that may be consequences of global change, including shifting currents; increased number and size of hypoxic or anoxic areas; and increased number and duration of harmful algal blooms. More information must be obtained through field and laboratory research and monitoring that involve innovative sampling techniques (such as genetics and acoustics), but data that already exist must be made accessible. And all data must have a temporal component so trends can be identified. As data are compiled, techniques must be developed to make certain that scales are compatible, to combine and reconcile data collected for various purposes with disparate gear, and to automate taxonomic changes. Information on biotic and abiotic elements of the environment must be interactively linked. Impediments to assembling existing data and collecting new data on marine biodiversity include logistical problems as well as shortages in finances and taxonomic expertise.     

Interaction of all-<Emphasis Type="Italic">trans</Emphasis>-Retinal with bilayer lipid membranes

The interaction of all-trans-retinal (hereinafter referred to as retinal) with planar bilayer lipid membranes has been studied. Addition of retinal into aqueous solutions on both sides of the membrane formed from diphytanoilphosphatidylcholine (DPhPC) or its mixture with diphytanoilphosphatidylethanolamine (DPhPC/DPhPE in w/w proportion of 3: 5) led to a change of conductance induced by ionophores nonactin (increase of conductance) or pentachlorophenol (decrease). Increase of nonactin-induced conductance was dependent on the membrane lipid composition and was two times higher in the case of DPhPC/DPhPE mixture. The change of conductance caused by ionophores of different signs (plus or minus) had different direction suggesting the influence of the retinal on the dipole potential upon its incorporation into BLM. The boundary potentials difference measured by the intramembrane field compensation method (IFC) after the retinal addition on one side of the membrane did not exceed 2.5 mV suggesting that its distribution in the bilayer is almost symmetrical. The illumination of the retinal-containing BLM caused a decrease in its lifetime when the membranes were formed from unsaturated lipids. Retinal incorporated into BLM led also to photoinactivation of the gramicidin channels. The process was completely inhibited by a singlet oxygen quencher (sodium azide). These results indicate that retinal accumulated in the membrane can affect both membrane proteins and the unsaturated lipids by their oxidation by the singlet oxygen.     

Comparative Genomic Analysis of Archaeal Genotypic Variants in a Single Population and in Two Different Oceanic Provinces

Planktonic crenarchaeotes are present in high abundance in Antarctic winter surface waters, and they also make up a large proportion of total cell numbers throughout deep ocean waters. To better characterize these uncultivated marine crenarchaeotes, we analyzed large genome fragments from individuals recovered from a single Antarctic picoplankton population and compared them to those from a representative obtained from deeper waters of the temperate North Pacific. Sequencing and analysis of the entire DNA insert from one Antarctic marine archaeon (fosmid 74A4) revealed differences in genome structure and content between Antarctic surface water and temperate deepwater archaea. Analysis of the predicted gene products encoded by the 74A4 sequence and those derived from a temperate, deepwater planktonic crenarchaeote (fosmid 4B7) revealed many typical archaeal proteins but also several proteins that so far have not been detected in archaea. The unique fraction of marine archaeal genes included, among others, those for a predicted RNA-binding protein of the bacterial cold shock family and a eukaryote-type Zn finger protein. Comparison of closely related archaea originating from a single population revealed significant genomic divergence that was not evident from 16S rRNA sequence variation. The data suggest that considerable functional diversity may exist within single populations of coexisting microbial strains, even those with identical 16S rRNA sequences. Our results also demonstrate that genomic approaches can provide high-resolution information relevant to microbial population genetics, ecology, and evolution, even for microbes that have not yet been cultivated.     

Parvalbumin in respiratory neurons of the ventrolateral medulla of the adult rat

A column of parvalbumin immunoreactive neurons is closely associated with the location of respiratory neurons in the ventrolateral medulla of the rat. The majority (66%) of bulbospinal neurons in the medullary ventral respiratory column (VRC) that were retrogradely labeled by tracer injections in the phrenic nucleus were also positive for parvalbumin. In contrast, only 18.8% of VRC neurons retrogradely labeled after a tracer injection in the VRC, also expressed parvalbumin. The average cross-sectional area of VRC neurons retrogradely labeled after VRC injections was 193.8 μm² ± 6.6 SE. These were significantly smaller than VRC parvalbumin neurons (271.9 μm² ± 12.3 SE). Parvalbumin neurons were found in the Bötzinger Complex, the rostral ventral respiratory group (VRG), and the caudal VRG, areas which all contribute to the bulbospinal projection. In contrast, parvalbumin neurons were sparse or absent in the preBötzinger Complex and in the vicinity of the retrotrapezoid nucleus, areas that have few bulbospinal projections. Parvalbumin was rarely colocalized within Neurokinin-1 receptor positive (NK1R) VRC neurons, which are found in the preBötzinger complex and in the anteroventral part of the rostral VRG. Parvalbumin neurons in the Bötzinger Complex and rostral VRG help define the rostrocaudal extent of these regions. The absence of parvalbumin neurons from the intervening preBötzinger complex also helps establish the boundaries of this region. Regional boundaries described in this manner are in good agreement with earlier physiological and anatomical studies. Taken together, the distributions of parvalbumin, NK1R and bulbospinal neurons suggest that the rostral VRG may be subdivided into distinct, anterodorsal, anteroventral, and posterior subdivisions.