An improved grafting technique for mature Arabidopsis plants demonstrates long-distance shoot-to-root transport of phytochelatins in Arabidopsis

Phytochelatins (PCs) are peptides that function in heavy-metal chelation and detoxification in plants and fungi. A recent study showed that PCs have the ability to undergo long-distance transport in a root-to-shoot direction in transgenic Arabidopsis (Arabidopsis thaliana). To determine whether long-distance transport of PCs can occur in the opposite direction, from shoots to roots, the wheat (Triticum aestivum) PC synthase (TaPCS1) gene was expressed under the control of a shoot-specific promoter (CAB2) in an Arabidopsis PC-deficient mutant, cad1-3 (CAB2TaPCS1/cad1-3). Analyses demonstrated that TaPCS1 is expressed only in shoots and that CAB2TaPCS1/cad1-3 lines complement the cadmium (Cd) and arsenic metal sensitivity of cad1-3 shoots. CAB2TaPCS1/cad1-3 plants exhibited higher Cd accumulation in roots and lower Cd accumulation in shoots compared to wild type. Fluorescence HPLC coupled to mass spectrometry analyses directly detected PC2 in the roots of CAB2:TaPCS1/cad1-3 but not in cad1-3 controls, suggesting that PC2 is transported over long distances in the shoot-to-root direction. In addition, wild-type shoot tissues were grafted onto PC synthase cad1-3 atpcs2-1 double loss-of-function mutant root tissues. An Arabidopsis grafting technique for mature plants was modified to obtain an 84% success rate, significantly greater than a previous rate of approximately 11%. Fluorescence HPLC-mass spectrometry showed the presence of PC2, PC3, and PC4 in the root tissue of grafts between wild-type shoots and cad1-3 atpcs2-1 double-mutant roots, demonstrating that PCs are transported over long distances from shoots to roots in Arabidopsis.     

Generation of transgenic plants expressing plasma membrane-bound antibodies to the environmental pollutant microcystin-LR

In this paper we describe the engineering and regeneration of transgenic tobacco plants expressing a recombinant plasma membrane-retained antibody specific to microcystin-LR (MC-LR), the environmental toxin pollutant produced by cyanobacteria. The antibody was created by a genetic fusion of the antigen binding regions of the microcystin-specific single chain antibody, 3A8, with the constant regions from the murine IgG1κ, Guy’s 13, including a membrane retention sequence at the C-terminal end of the antibody heavy chain. The antibody produced in the leaves was shown to be functional by binding to MC-LR in an ELISA with antibody yields in transgenic plant leaves reaching a maximum of 1.2 μg g⁻¹ leaf f.wt (0.005% total soluble protein). Antibody-MC-LR complexes formed in leaves after addition of MC-LR to hydroponic medium around the roots of transgenic plant cultures.     

Phylogeographic insights into the invasion history and secondary spread of the signal crayfish in Japan

Successful invasion by nonindigenous species is often attributed to high propagule pressure, yet some foreign species become widespread despite showing reduced genetic variation due to founder effects. The signal crayfish (Pacifastacus leniusculus) is one such example, where rapid spread across Japan in recent decades is believed to be the result of only three founding populations. To infer the history and explore the success of this remarkable crayfish invasion, we combined detailed phylogeographical and morphological analyses conducted in both the introduced and native ranges. We sequenced 16S mitochondrial DNA of signal crayfish from across the introduced range in Japan (537 samples, 20 sites) and the native range in western North America (700 samples, 50 sites). Because chela size is often related to aggressive behavior in crayfish, and hence, their invasion success, we also measured chela size of a subset of specimens in both introduced and native ranges. Genetic diversity of introduced signal crayfish populations was as high as that of the dominant phylogeographic group in the native range, suggesting high propagule pressure during invasion. More recently established crayfish populations in Japan that originated through secondary spread from one of the founding populations exhibit reduced genetic diversity relative to older populations, probably as a result of founder effects. However, these newer populations also show larger chela size, consistent with expectations of rapid adaptations or phenotypic responses during the invasion process. Introduced signal crayfish populations in Japan originate from multiple source populations from a wide geographic range in the native range of western North America. A combination of high genetic diversity, especially for older populations in the invasive range, and rapid adaptation to colonization, manifested as larger chela in recent invasions, likely contribute to invasion success of signal crayfish in Japan.     

Identification of peptidases in Nicotiana tabacum leaf intercellular fluid

Peptidases in the extracellular space might affect the integrity of recombinant proteins expressed in, and secreted from, plant cells. To identify extracellular peptidases, we recovered the leaf intercellular fluid from Nicotiana tabacum plants by an infiltration-centrifugation method. The activity of various peptidases was detected by an in vitro assay in the presence of specific inhibitors, using BSA and human serum gamma-globulin as substrates. Peptidases were detected by 1- and 2-D zymography in a polyacrylamide gel containing gelatin as substrate. Proteolytic activity was observed over a wide range of molecular masses equal to, or higher than, 45 kDa. To identify the peptidases, the extracellular proteins were digested with trypsin and analyzed by LC and MS. Seventeen peptides showing identity or similarity to predicted plant aspartic, cysteine, and serine peptidases have been identified. The extracellular localization of a cysteine peptidase aleurain homolog was also shown.     

Structure and permeability of the egg capsule of the bonnethead shark, Sphyrna tiburo

In the viviparous bonnethead shark, Sphyrna tiburo, a fluid-filled, acellular egg capsule surrounds fertilized eggs and developing embryos throughout gestation. Like other placental shark species, the capsule remains intact even at the placental implantation site. Although its intervention between the uterine and embryonic tissues of the placenta has long been thought to mediate physiological exchange, little information is available concerning even its basic structure or permeability to solutes. The 1 mum thick capsule wall consists of an inner layer of gelatinous material and an outer layer consisting of at least three laminae of orthogonally arranged fibrous material. These fibers are irregular and often branched. Permeability experiments showed that solutes less than 1,355 Da diffuse across the egg capsule whereas those greater than 6,000 Da do not pass through the membrane. Solute movement across the capsule is a concentration-dependent phenomenon indicating diffusion rather than active transport. Experimental data also suggest that there is an increase in the permeability of the egg capsule to low molecular weight materials during mid- and late gestation. These observations are discussed in relation to the function of the egg capsule as a mediator of maternal-embryonic interactions in matrotrophic sharks.     

Construction of a sequencedClostridium perfringens-Escherichia coli shuttle plasmid

A newClostridium perfringens-Escherichia coli shuttle plasmid has been constructed and its complete DNA sequence compiled. The vector, pJIR418, contains the replication regions from theC. perfringens replicon pIP404 and theE. coli vector pUC18. The multiple cloning site and lacZ gene from pUC18 are also present, which means that X-gal screening can be used to select recombinants inE. coli. Both chloramphenicol and erythromycin resistance can be selected inC. perfringens andE. coli since pJIR418 carries theC. perfringens catP and ermBP genes. Insertional inactivation of either the catP or ermBP genes can also be used to directly screen recombinants in both organisms. The versatility of pJIR418 and its applicability for the cloning of toxin genes fromC. perfringens have been demonstrated by the manipulation of a cloned gene encoding the production of phospholipase C.     

Efficacy of several insecticides alone and with horticultural mineral oils on light brown apple moth (Lepidoptera: Tortricidae) eggs

The aim of the research was to identify efficacious and less environmentally harmful treatments than the standard chlorpyrifos sprays used for the control light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), eggs on nursery stock. A series of dip experiments showed a range of responses when comparing the efficacy of insecticides on egg hatch of E. postvittana. The insecticides that compared most favorably with chlorpyrifos were lamda-cyhalothrin and gamma-cyhalothrin, and thiacloprid. Indoxacarb, novaluron, and spinosad caused significant mortality only when combined with All Seasons mineral oil. All Seasons, showed ovicidal properties when evaluated alone and demonstrated adjuvant properties when combined with the above-mentioned insecticides, except gamma-cyhalothrin and thiacloprid. Several other horticultural mineral oils performed similarly, except the efficacy of spinosad varied with the oil product used, suggesting that the oil type selected is important for some insecticide and oil combinations. Several insecticides evaluated in this study are likely candidates for further work to develop treatments against E. postvittana eggs on nursery plants. Mineral oils are ovicidal and combinations with insecticides are likely to be advantageous.     

Assignment of the proton NMR spectrum of reduced and oxidized thioredoxin: sequence-specific assignments, secondary structure, and global fold

Complete proton assignments are reported for the 1H nuclear magnetic resonance (NMR) spectrum of Escherichia coli thioredoxin in the oxidized (with active-site disulfide bridge) and reduced (with two sulfhydryl groups) states. The assignments were obtained by using an integrated assignment strategy in which spin systems were identified from a combination of relayed and multiple quantum NMR techniques prior to sequential assignment. Elements of secondary structure were identified in each protein from characteristic nuclear Overhauser effects (NOE), coupling constants, and slowly exchanging amide protons. In both oxidized and reduced thioredoxin, approximately 33% of the 108 amino acid residues participate in a beta-sheet containing four major strands (three antiparallel and one parallel). A further short beta-strand is connected in a parallel fashion at the N-terminal end of the sheet. Two of the antiparallel beta-strands are connected by a 7-residue beta-bulge loop. Three helical segments, also containing approximately 33% of the amino acid residues, are well-defined in both oxidized and reduced thioredoxin. The remaining third of the molecule apparently consists of reverse turns and loops with little defined secondary structure. The global folds of oxidized and reduced thioredoxin are shown to be essentially identical. Both NOE connectivities and chemical shift values for the two proteins are very similar, except in the immediate vicinity of the active site where significant variations in the chemical shift indicate subtle conformational changes. While the overall fold of oxidized thioredoxin is the same in solution and in the crystalline state, some small differences in local conformation are apparent.     

Consequences of Lineage-Specific Gene Loss on Functional Evolution of Surviving Paralogs: ALDH1A and Retinoic Acid Signaling in Vertebrate Genomes

Genome duplications increase genetic diversity and may facilitate the evolution of gene subfunctions. Little attention, however, has focused on the evolutionary impact of lineage-specific gene loss. Here, we show that identifying lineage-specific gene loss after genome duplication is important for understanding the evolution of gene subfunctions in surviving paralogs and for improving functional connectivity among human and model organism genomes. We examine the general principles of gene loss following duplication, coupled with expression analysis of the retinaldehyde dehydrogenase Aldh1a gene family during retinoic acid signaling in eye development as a case study. Humans have three ALDH1A genes, but teleosts have just one or two. We used comparative genomics and conserved syntenies to identify loss of ohnologs (paralogs derived from genome duplication) and to clarify uncertain phylogenies. Analysis showed that Aldh1a1 and Aldh1a2 form a clade that is sister to Aldh1a3-related genes. Genome comparisons showed secondarily loss of aldh1a1 in teleosts, revealing that Aldh1a1 is not a tetrapod innovation and that aldh1a3 was recently lost in medaka, making it the first known vertebrate with a single aldh1a gene. Interestingly, results revealed asymmetric distribution of surviving ohnologs between co-orthologous teleost chromosome segments, suggesting that local genome architecture can influence ohnolog survival. We propose a model that reconstructs the chromosomal history of the Aldh1a family in the ancestral vertebrate genome, coupled with the evolution of gene functions in surviving Aldh1a ohnologs after R1, R2, and R3 genome duplications. Results provide evidence for early subfunctionalization and late subfunction-partitioning and suggest a mechanistic model based on altered regulation leading to heterochronic gene expression to explain the acquisition or modification of subfunctions by surviving ohnologs that preserve unaltered ancestral developmental programs in the face of gene loss.