Acquired resistance to the rice blast in transgenic rice accumulating the antimicrobial peptide thanatin

Thanatin is an antimicrobial peptide with a strong and wide-ranging antimicrobial spectrum, including certain species of fungi and Gram-negative and Gram-positive bacteria. To evaluate the application of thanatin to the generation of disease-resistant plants, we introduced a synthetic thanatin gene into rice. Several transformants that expressed the introduced gene showed significant level of antimicrobial activity. The substances showing antimicrobial activity were partially purified from these transformants and their properties were determined. The molecule with characteristics similar to those of native thanatin on the elution pattern in HPLC analysis had an identical molecular mass to that of native molecule. It should also be noted that the transformant acquired a sufficient level of resistance to the rice blast fungus, Magnaporthe oryzae, presumably due to the repressive activity of thanatin to its initial stage of infection. This result demonstrates that thanatin has antifungal activity for M. oryzae and that the introduction of the thanatin gene into rice is effective in generating a plant resistant to rice blast disease.     

Quantitative analysis of plant polyamines including thermospermine during growth and salinity stress

Arabidopsis thaliana was thought to contain two spermine synthase genes, ACAULIS 5 (ACL5) and SPMS. Recent investigations, however, revealed that the ACL5 gene encodes thermospermine synthase. In this study, we have established a simple method to separate two isomers of tetraamine, spermine and thermospermine, in extracts from plant tissues of less than 500 mg. Polyamines (PAs) extracted from plant tissues were benzoylated, and the derivatives were completely resolved by high-performance liquid chromatography on a C₁₈ reverse-phase column, by eluting with 42% (v/v) acetonitrile in water in an isocratic manner at 30 °C and monitoring at 254 nm. The relevance of the method was confirmed by co-chromatography with respective PAs and by the PA analysis of the single- and double-mutants of acl5 and spms, which could not synthesize thermospermine and/or spermine, respectively. Furthermore, with this method, we monitored the thermospermine contents in various tissues of A. thaliana and found that stems and flowers contain two- to three-fold more thermospermine compared to whole seedlings and mature leaves. The presence of thermospermine was confirmed in Oryza sativa and Lycopersicon pesculentum. Finally we addressed whether salinity stress changes the contents of PAs including thermospermine in Arabidopsis.     

CLASPs attach microtubule plus ends to the cell cortex through a complex with LL5beta

CLASPs are mammalian microtubule-stabilizing proteins that can mediate the interaction between distal microtubule ends and the cell cortex. Using mass spectrometry-based assays, we have identified two CLASP partners, LL5beta and ELKS. LL5beta and ELKS form a complex that colocalizes with CLASPs at the cortex of HeLa cells as well as at the leading edge of motile fibroblasts. LL5beta is required for cortical CLASP accumulation and microtubule stabilization in HeLa cells, while ELKS plays an accessory role in these processes. LL5beta is a phosphatidylinositol-3,4,5-triphosphate (PIP3) binding protein, and its recruitment to the cell cortex is influenced by PI3 kinase activity but does not require intact microtubules. Cortical clusters of LL5beta and ELKS do not overlap with focal adhesions but often form in their vicinity and can affect their size. We propose that LL5beta and ELKS can form a PIP3-regulated cortical platform to which CLASPs attach distal microtubule ends.     

Novel Cysteine-Rich Peptides from Digitaria ciliaris and Oryza sativa Enhance Tolerance to Cadmium by Limiting its Cellular Accumulation

By means of functional screening using the cadmium (Cd)-sensitiveycf1 yeast mutant, we have isolated a novel cDNA clone, DcCDT1,from Digitaria ciliaris growing in a former mining area in northernJapan, and have shown that it confers Cd tolerance to the yeastcells, which accumulated almost 2-fold lower Cd levels thancontrol cells. The 521 bp DcCDT1 cDNA contains an open readingframe of 168 bp and encodes a deduced peptide, DcCDT1, thatis 55 amino acid residues in length, of which 15 (27.3%) arecysteine residues. Five DcCDT1 homologs (here termed OsCDT1–OsCDT5)have been identified in rice, and all of them were up-regulatedto varying degrees in the above-ground tissues by CdCl₂ treatment.Localization of green fluorescent protein fusions suggests thatDcCDT1 and OsCDT1 are targeted to both cytoplasmic membranesand cell walls of plant cells. Transgenic Arabidopsis thalianaplants overexpressing DcCDT1 or OsCDT1 displayed a Cd-tolerantphenotype and, consistent with our yeast data, accumulated loweramounts of Cd when grown on CdCl₂. Collectively, our data suggestthat DcCDT1 and OsCDT1 function to prevent entry of Cd intoyeast and plant cells and thereby enhance their Cd tolerance.     

Carbohydrate structural analysis of wheat flour arabinogalactan protein

The water-extractable arabinogalactan protein (AGP) was isolated from bread wheat flour (Triticum aestivum L. variety Cadenza) and the structure of the arabinogalactan (AG) carbohydrate component was studied. Oligosaccharides, released by hydrolysis of the AG with a range of AGP-specific enzymes, were characterised by Matrix Assisted Laser Desorption Ionisation (MALDI)-Time of Flight (ToF)-Mass Spectrometry (MS), MALDI-ToF/ToF high energy collision induced dissociation (CID) and Polysaccharide Analysis by Carbohydrate gel Electrophoresis (PACE). The AG is composed of a β-(1→3)-d-galactan backbone with β-(1→6)-d-galactan side chains. These side chains are highly variable in length, from one to at least 20 Gal residues and are highly substituted with α-l-Araf. Single GlcA residues are also present at the non-reducing termini of some short β-(1→6)-galactan side chains. In addition, the β-(1→6)-galactan side chains are also substituted with β-l-Arap. We propose a polysaccharide structure of the wheat flour AGP that is substantially revised from earlier models.     

Synthesis of N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl ester catalyzed by thermolysin variants with improved activity

Thermolysin is industrially used for the synthesis of N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl ester (ZDFM), a precursor of an artificial sweetener, aspartame, from N-carbobenzoxy-l-aspartic acid (ZD) and l-phenylalanine methyl ester (FM). We have reported five thermolysin variants [D150A (Asp150 is replaced with Ala), D150E, D150W, I168A, and N227H] with improved activity generated by site-directed mutagenesis of the residues located at the active site [Kusano et al. J Biochem 2009;145:103–13]. In this study, we analyzed the ZDFM synthesis reaction catalyzed by these variants. Steady-state kinetic analysis revealed that in the ZDFM synthesis reaction at pH 7.5, at 25 °C, the molecular activity k_cat values of the variants were 1.6–3.8 times higher than that of the wild-type thermolysin (WT), while their Michaelis constant K_m values for ZD and FM were almost the same as those of WT. With the initial concentrations of enzyme, ZD, and FM of 0.1 μM, 5 mM, and 5 mM, respectively, the synthesis of ZDFM catalyzed by these variants reached the maximum level at 4 h while that catalyzed by WT did at 12 h. These results suggest that the five thermolysin variants examined are more suitable than WT for use in ZDFM synthesis.     

Possible mapping of the gene for transient myeloproliferative syndrome at 21q11.2

Summary The parental origin of the extra chromosome 21 was studied in 20 patients with trisomy 21-associated transient myeloproliferative syndrome (TMS) using chromosomal heteromorphisms as markers; this was combined with a study of DNA polymorphisms in 5 patients. Of these, 10 were shown to result from duplication of a parental chromosome 21, viz., maternal in 8 and paternal in 2. A patient with Down syndrome-associated TMS had a paracentric inversion in two of his three chromosomes 21 [47,XY,-21, +inv(21)(q11.2q22.13)mat, +inv(21)(q11.2 q22.13)mat). These findings support our hypothesis of disomic homozygosity of a mutant gene on chromosome 21 in 21-trisomic cells as being a mechanism responsible for the occurrence of TMS. The finding also suggests that the putative TMS gene locus is at either 21q11.2 or 21q22.13, assuming that the gene is interrupted at either site because of the inversion. The study of 5 TMS patients using DNA polymorphic markers detected a cross-over site on the duplicated chromosomes 21 between 21q11.2 (or q21.2) and 21q21.3 in one patient, and a site between 21q21.3 and q22.3 in another patient, evidence that confined the gene locus to the 21cen-q21.3 segment. These findings suggest that the putative TMS gene is located at 21q11.2. The extra chromosome 21 in the latter two TMS patients probably resulted from maternal second meiotic non-disjunction, in view of the presence of recombinant heterozygous segments on their duplicated chromosomes 21.