The Pathway and Regulation of Salicylic Acid Biosynthesis in Probenazole-Treated <Emphasis Type="Italic">Arabidopsis</Emphasis>

Probenazole (PBZ; 3-allyloxy-1,2-benzisothiazole-1,1-dioxide) is a highly effective chemical inducer of systemic-acquired resistance (SAR). It has been used widely to protect rice plants against the rice blast fungus Magnaporthe grisea. Previous studies have shown that PBZ induces SAR through enhanced accumulation of salicylic acid (SA). Plants synthesize SA by either a pathway that uses phenylalanine as substrate or another that involves isochorismate. To clarify how SA is produced in PBZ-treated Arabidopsis, we examined the expression patterns and enzyme activities of phenylalanine ammonia lyase (PAL) and isochorismate synthase (ICS), which are the main components of the phenylalanine and isochorismate pathways, respectively. PBZ exposure significantly improved the accumulation of SA and increased ICS activity. In the sid2–2 mutant, which has a defect in ICS1, PBZ had no effect on the level of endogenous SA or activity of ICS. In contrast, PAL activity and the expression of most PAL genes were down-regulated by such treatment in wild-type plants. These results suggest that SA is mainly synthesized via the ICS-mediated pathway in Arabidopsis.     

A Pleiotropic Phenotype is Associated with Altered Endogenous Hormone Balance in the Developmentally Stunted Mutant (<Emphasis Type="Italic">dsm1</Emphasis>)

A developmentally stunted mutant (dsm1) of Arabidopsis, isolated from an EMS mutant screen, had a pleiotropic phenotype, including repressed germination, retarded growth, delayed flowering, and impaired fertility. Additionally, dsm1 had a lifespan of approximately 160 days, which was more than twice the lifespan of the wild type (Col-0). Fine morphological and anatomical characters, such as the shoot apical meristem, root apical meristem, seed shape, and seed surface, were obviously altered in dsm1. We found that both abscisic acid and zeatin riboside levels were significantly greater in dsm1 than in Col-0 at all stages of development, while the levels of indole-3-acetic acid and gibberellins varied by age. The expressions of some abscisic acid-related genes were higher in dsm1 than in Col-0. These data indicate that DSM1 may play a general role in plant growth and development.     

Exploring novel bioactive compounds from marine microbes

The historical paradigm of the deep ocean as a biological 'desert' has shifted to one of a 'rainforest' owing to the isolation of many novel microbes and their associated bioactive compounds. Recently, there has been an explosion of information about novel bioactive compounds that have been isolated from marine microbes in an effort to further explore the relatively untapped marine microbes and their secondary metabolites for drug discovery. The microbes are recovered and purified from the ocean by both conventional and innovative isolation methods to obtain those previously thought to be 'uncultivable'. To overcome the difficulties and limitations associated with cultivation techniques, several DNA-based molecular methods have been developed to bypass the culture-dependent bottleneck. Bioactive compounds isolated using the above strategies have not only shown importance in biotechnological and pharmaceutical applications but have also increased our understanding of the diversity of marine microbiota, ecosystem functions and the exploitable biology.     

Screening for stable transformants and stability of β-glucuronidase gene expression in suspension cultured cells of tall fescue (Festuca arundinacea)

Summary By screening cell colonies derived from protoplasts of tall fescue (Festuca arundinacea), transformed with a rice actin-1-promoter-ß-glucuronidase gene construct, several ß-glucuronidase positive callus clones were obtained. Two callus clones with different GUS expression were derived from these. One was light blue after X-gluc staining, and expression of the ß-glucuronidase gene was stable over repeated subculture, while another stained intensely blue, and expression of the ß-glucuronidase gene was unstable. Southern blot analysis showed that only one copy of the ß-glucuronidase gene was integrated into the genome, and that these two clones appeared to have the same integration pattern. Treatment with 5-azacytidine maintained GUS expression in the unstable line but had no effect on reactivating expression of the GUS gene after expression had been lost. Following the screening procedure the callus clones would only regenerate albino plants.Abbreviations X-gluc 5-bromo-4-chloro-3-indolylglucuronide - GUS ß-glucuronidase - CaMV cauliflower mosaic virus - PEG polyethylene glycol - AZC 5-azacytidine - SDS sodium dodecyl sulphate - UV ultraviolet - EDTA ethylenediaminetetra-acetic acid disodium salt - SSPE salt-sodium-phosphate-EDTA - SSC standard saline citrate - hpt hygromycin phosphotransferase     

Regulation of Bestrophin Cl Channels by Calcium: Role of the C Terminus

Human bestrophin-1 (hBest1), which is genetically linked to several kinds of retinopathy and macular degeneration in both humans and dogs, is the founding member of a family of Cl⁻ ion channels that are activated by intracellular Ca²⁺. At present, the structures and mechanisms responsible for Ca²⁺ sensing remain unknown. Here, we have used a combination of molecular modeling, density functional–binding energy calculations, mutagenesis, and patch clamp to identify the regions of hBest1 involved in Ca²⁺ sensing. We identified a cluster of a five contiguous acidic amino acids in the C terminus immediately after the last transmembrane domain, followed by an EF hand and another regulatory domain that are essential for Ca²⁺ sensing by hBest1. The cluster of five amino acids (293–308) is crucial for normal channel gating by Ca²⁺ because all but two of the 35 mutations we made in this region rendered the channel incapable of being activated by Ca²⁺. Using homology models built on the crystal structure of calmodulin (CaM), an EF hand (EF1) was identified in hBest1. EF1 was predicted to bind Ca²⁺ with a slightly higher affinity than the third EF hand of CaM and lower affinity than the second EF hand of troponin C. As predicted by the model, the D312G mutation in the putative Ca²⁺-binding loop (312–323) reduced the apparent Ca²⁺ affinity by 20-fold. In addition, the D312G and D323N mutations abolished Ca²⁺-dependent rundown of the current. Furthermore, analysis of truncation mutants of hBest1 identified a domain adjacent to EF1 that is rich in acidic amino acids (350–390) that is required for Ca²⁺ activation and plays a role in current rundown. These experiments identify a region of hBest1 (312–323) that is involved in the gating of hBest1 by Ca²⁺ and suggest a model in which Ca²⁺ binding to EF1 activates the channel in a process that requires the acidic domain (293–308) and another regulatory domain (350–390). Many of the ~100 disease-causing mutations in hBest1 are located in this region that we have implicated in Ca²⁺ sensing, suggesting that these mutations disrupt hBest1 channel gating by Ca²⁺.     

Transformation of oat and inheritance of bar gene expression

Fertile transgenic plants of oat (Avena sativa L. var. Melys) were produced following microprojectile bombardment of primary embryogenic calli from immature embryos with two plasmids containing the bar gene or the β-glucuronidase (uidA) gene, after selection with glufosinate ammonium. Eleven plants were regenerated from phosphinothricin resistant callus, with three of the eleven plants containing either intact or rearranged copies. No plants co-transformed with the non-selected uidA gene were detected. Stable transmission and expression of the bar gene in the T₁ inbred progenies occurred in a Mendelian manner in one line, which contained an intact bar gene, and in all six T₂ lines tested from this transformant. This revised version was published online in June 2006 with corrections to the Cover Date.     

How to map ses, a mutant of Arabidopsis thaliana affecting pollen development

In Arabidopsis, map-based cloning has been developed to an effective method in mutant genetic analysis because high-density markers are available, candidate genes or genomic sequences can be amplified by PCR, and transgenic techniques are simplified. Mutant ses named from shortened early-stage siliques was used as an example to show how to map a mutant in this way. By the process of bulked segregants analysis, linkage testing, large-scale and fine-scale mapping, mutant ses was narrowed into a 67 kb interval from CER448792 (2000541 bp) to CER464544 (2067844 bp) crossing over the right of BAC F12K11 to the left of the BAC F4H5 including at most 22 putative genes on the top of chromosome 1. In sequence-based map of Arabidopsis genes with mutant phenotype (SMAGMP) mutant ses was between AT1g06150 (EMB1444) and AT1g08060 (MOM). The ses mapping also showed that developed markers on polymorphism site of CAPC not only were simplified but worked well. Twenty-four markers from CAPC used in the mapping maybe help Arabidopsis researchs with others and the methods related to ses mapping also gave an example of positional cloning. The text was submitted by the authors in English.     

Protein interaction data set highlighted with human Ras-MAPK/PI3K signaling pathways

The Ras-MAPK and PI3K-AKT pathways are conserved in metazoan organisms, which involve a series of signaling cascades and form the basis for numerous physiological and pathological processes. Here we report on yeast two hybrid screening results of a protein interaction network around the known components of human Ras-MAPK/PI3K pathways. A total of 42 independent cDNA library screenings resulted in 200 protein-protein interaction (PPI) pairs among 180 molecules. Most of the proteins formed a large cluster that contains 193 PPIs between 169 proteins. Seventy-four interactions indicate high-confidence according to bioinformatics analysis. The prey list contains high enrichment genes with specific Gene Ontology (GO) terms such as response to stress and response to external stimulus. Most interactions link the Ras signaling pathway with various cellular processes. Five interactions were validated by coimmunoprecipitation and colocalization assays in mammalian cells to confirm their in vivo interactions. This protein interaction network provides further insights into the molecular mechanism of Ras-MAPK/PI3K signaling pathways.     

PatternLab for proteomics: a tool for differential shotgun proteomics

Background  

A goal of proteomics is to distinguish between states of a biological system by identifying protein expression differences. Liu et al. demonstrated a method to perform semi-relative protein quantitation in shotgun proteomics data by correlating the number of tandem mass spectra obtained for each protein, or "spectral count", with its abundance in a mixture; however, two issues have remained open: how to normalize spectral counting data and how to efficiently pinpoint differences between profiles. Moreover, Chen et al. recently showed how to increase the number of identified proteins in shotgun proteomics by analyzing samples with different MS-compatible detergents while performing proteolytic digestion. The latter introduced new challenges as seen from the data analysis perspective, since replicate readings are not acquired.