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Accumulation of dehydrin-like proteins in the mitochondria of cereals in response to cold, freezing, drought and ABA treatment.

BACKGROUND: Dehydrins are known as Group II late embryogenesis abundant proteins. Their high hydrophilicity and thermostability suggest that they may be structure stabilizers with detergent and chaperone-like properties. They are localised in the nucleus, cytoplasm, and plasma membrane. We have recently found putative dehydrins in the mitochondria of some cereals in response to cold. It is not known whether dehydrin-like proteins accumulate in plant mitochondria in response to stimuli other than cold stress. RESULTS: We have found five putative dehydrins in the mitochondria of winter wheat, rye and maize seedlings. Two of these polypeptides had the same molecular masses in all three species (63 and 52 kD) and were thermostable. Drought, freezing, cold, and exogenous ABA treatment led to higher accumulation of dehydrin-like protein (dlp) 63 kD in the rye and wheat mitochondria. Protein 52 kD was induced by cold adaptation and ABA. Some accumulation of these proteins in the maize mitochondria was found after cold exposition only. The other three proteins appeared to be heat-sensitive and were either slightly induced or not induced at all by all treatments used. CONCLUSIONS: We have found that, not only cold, but also drought, freezing and exogenous ABA treatment result in accumulation of the thermostable dehydrins in plant mitochondria. Most cryotolerant species such as wheat and rye accumulate more heat-stable dehydrins than cryosensitive species such as maize. It has been supposed that their function is to stabilize proteins in the membrane or in the matrix. Heat-sensitive putative dehydrins probably are not involved in the stress reaction and adaptation of plants.

Abscisic Acid↗

Lignan precursors from flaxseed or rye bran do not protect against the development of intestinal neoplasia in ApcMin mice.

Phytoestrogens, like isoflavonoids and lignans, have been postulated as possible colorectal cancer protective constituents. To investigate this hypothesis, two high-fiber sources rich in lignan precursors, i.e., rye bran and flaxseed, were tested for their ability to modulate intestinal tumor development in ApcMin mice. Test diets consisted of a control diet (a Western-style diet, adjusted for fiber and/or phytate content) supplemented with 5% flaxseed or 30% rye bran. Chemical analysis of diets and blood samples confirmed the enhanced systemic exposure of mice fed the test diets to the major lignan precursors, i.e., secoisolariciresinol and matairesinol. No statistically significant difference was observed in the incidence and multiplicity of small intestinal and colon tumors at terminal sacrifice between mice fed the control diet or the diet supplemented with 5% flaxseed. With the rye bran diet a statistically significant enhancement of the number of small intestinal tumors in female mice was observed. The number of colon tumors, however, was comparable between the control and rye bran-fed mice of either sex. Furthermore, no activating point mutations in the K-ras oncogene nor positive immunohistochemical staining for the p53 gene were observed in a set of 48 colon tumors. In conclusion, our results demonstrate that increased intake of lignan precursors from flaxseed or rye bran, administered in a Western-style diet, does not protect against intestinal tumor development in an appropriate animal model for intestinal neoplasia such as the ApcMin mice.

Animals↗

Rye bran diet increases epithelial cell apoptosis and decreases epithelial cell volume in TRAMP (transgenic adenocarcinoma of the mouse prostate) tumors.

A rye bran diet has been suggested to retard prostate cancer growth and increase tumor cell apoptosis in animal models and in men with prostate cancer. Transgenic mice designed to develop prostate cancer (transgenic adenocarcinoma of the mouse prostate, TRAMP) were therefore treated with a rye bran diet. TRAMP mice were fed a rye bran (n = 13) or a control diet (n = 15) from 4 to 24 wk of age. Tumor histology, cell proliferation, and apoptosis were accessed using stereological and immunohistochemical techniques. In the dorsolateral prostate lobe in TRAMP mice, epithelial cell apoptosis was increased by 31%, and the volume fraction of epithelial cells was reduced by 20% (P < 0.05) in the rye bran-treated group compared with the control diet group. There were, however, no significant changes detected in lobe weights or tumor grade. In conclusion, a rye bran diet increases epithelial apoptosis and reduces the epithelial cell fraction in TRAMP tumors.

Adenocarcinoma↗

Reorganization and polarization of the meiotic bouquet-stage cell can be uncoupled from telomere clustering.

Striking cellular reorganizations mark homologous pairing during meiotic prophase. We address the interdependence of chromosomal and cellular polarization during meiotic telomere clustering, the defining feature of the bouquet stage, by examining nuclear positioning and microtubule and nuclear pore reorganization. Polarization of meiotic cellular architecture was coincident with telomere clustering: microtubules were focused on the nuclear surface opposite the telomere cluster, the nucleus was positioned eccentrically in the cell such that the telomeres faced the direction of nuclear displacement and nuclear pores were clustered in a single region of the nuclear surface opposite the telomeres. Treatment of pre-bouquet stage cells with colchicine inhibited telomere clustering. Asymmetric nuclear positioning and nuclear pore clustering were normal in the presence of unclustered telomeres resulting from colchicine treatment. Nuclear pores were positioned normally with respect to the cell cortex in the absence of telomere clustering, indicating that telomere positioning is not required for polarization. This work provides evidence of meiotic cell polarization and suggests that telomeres may be positioned relative to an asymmetry present in the cell at the time of bouquet formation.

Cell Nucleus↗

Meiotic telomere clustering is inhibited by colchicine but does not require cytoplasmic microtubules.

Telomere clustering, the defining feature of the bouquet, is an almost universal feature of meiotic prophase, yet its mechanism remains unknown. The microtubule-depolymerizing agent colchicine was found to inhibit bouquet formation. Telomeres in colchicine-treated cells remained scattered in the nuclear periphery, whereas untreated cells exhibited a prominent telomere cluster. Colchicine administered after the bouquet had formed did not affect telomere dispersal. The effect of colchicine on bouquet formation appeared to be separable from its effect on cytoplasmic microtubules; amiprophos methyl, a highly effective plant microtubule-depolymerizing drug, did not affect telomere clustering. Inhibition of bouquet formation was limited to colchicine and the related drug podophyllotoxin out of the variety of microtubule-depolymerizing drugs tested, suggesting that the target involved in bouquet formation has a structural specificity.

Cells, Cultured↗

5-Methylcytosine distribution and genome organization in triticale before and after treatment with 5-azacytidine.

Triticale (2n=6x=42) is a hybrid plant including rye (R) and wheat (A and B) genomes. Using genomic in situ hybridization with rye DNA as a probe, we found the chromosomes of the R genome were not intermixed with the wheat chromosomes in 85% of nuclei. After treatment of seedlings with low doses of the drug 5-azacytidine (5-AC), leading to hypomethylation of the DNA, the chromosomes became intermixed in 60% of nuclei; the next generation showed intermediate organization. These results correlate with previous data showing that expression of R-genome rRNA genes, normally suppressed, is activated by 5-AC treatment and remains partially activated in the next generation. The distribution of 5-methylcytosine (5-mC) was studied using an antibody to 5-mC. Methylation was detected along the lengths of all chromosomes; there were some chromosome regions with enhanced and reduced methylation, but these were not located at consistent positions, nor were there differences between R and wheat genome chromosomes. After 5-AC treatment, lower levels of methylation were detected. After 5-AC treatment, in situ hybridization with rye genomic DNA sometimes showed micronuclei of rye origin and multiple translocations between wheat and rye chromosomes. Genomic DNA was analysed using methylation-sensitive restriction enzymes and, as probes, two rDNA sequences, two tandemly organised DNA sequences from rye (pSc200 and pSc250), and copia and the gypsy group retrotransposon fragments from rye and wheat. DNA extracted immediately after 5-AC treatment was cut more by methylation-sensitive restriction enzymes than DNA from untreated seedlings. Each probe gave a characteristic restriction fragment pattern, but rye- and wheat-origin probes behaved similarly, indicating that hypomethylation was induced in both genomes. In DNA samples from leaves taken 13-41 days after treatment, RFLP (Restriction Fragment Length Polymorphism) patterns were indistinguishable from controls and 5-AC treatments with all probes. Surprising differences in hybridization patterns were seen between DNA from root tips and leaves with the copia-fragment probes.

5-Methylcytosine↗

Genome restructuring in rye affects the expression, organization and disposition of homologous rDNA loci.

The standard rye cultivar 'Imperial' and a structural variant carrying an intact 1R chromosome and two telocentric 1R chromosomes (short and long arms) were used to investigate expression patterns of homologous rDNA loci, and the influence of chromosome structural change on their interphase organisation and relative disposition. Sequential silver staining and in situ hybridization with the rDNA probe pTa71, established a correspondence between the expression and organization patterns of rDNA domains in metaphase and interphase cells. In most cells of the cultivar Imperial, nucleolar organizer region (NOR) silver staining on metaphase chromosomes with equivalent numbers of rDNA genes revealed a size heteromorphism between homologous rDNA loci, resulting from their differential expression. NOR heteromorphism in the structural variant line was significantly reduced. The preferential activity of one NOR over its homologue was found to be random within cells and independent of parental origin. Nucleotypic modifications mediated by changes in the 1R chromosome structure include increased proximity between homologous rDNA loci in interphase, and an increase in the frequency of cells with intra-nucleolar ribosomal condensed chromatin. These results seem to indicate a 'sequence recognition' process for the regulation of homologous loci.

Cell Cycle↗

[Inhibitory effect and synergism of cernitin pollen extract on the urethral smooth muscle and diaphragm of the rat].

Inhibitory effects of cernitin pollen extract (CN-009), consisting of T-60 (a water-soluble extract) and GBX (an acetone-soluble extract) at a ratio of 20:1, were investigated in rat urethral smooth muscle and diaphragm. In the urethral smooth muscle, CN-009 (3.0 x 10(-4) approximately 3.0 x 10(-3) g/ml), T-60 and GBX (corresponding to CN-009) concentration-dependently inhibited the noradrenaline (NA, 10(-5) g/ml)-induced contraction. According to Bürgi's calculation, the inhibition by T-60 and GBX was synergistic. On the other hand, GBX inhibited the NA-contraction non-competitively and also inhibited the K+-contraction. In contrast, T-60 tended to inhibit competitively, but did not affect the K+-contraction. In the diaphragm, CN-009 (5.25 x 10(-3) approximately 2.1 x 10(-2) g/ml) concentration-dependently inhibited the nerve stimulation-induced twitch response. T-60 (corresponding to CN-009) showed no effect, while GBX slightly inhibited the twitch response. The effects of T-60 and GBX were synergistic. The inhibitory effects of CN-009 (2.1 x 10(-2) g/ml) and GBX (1.0 x 10(-2) g/ml) were specific against the nerve stimulation and were not antagonized by neostigmine (1.0 x 10(-5) g/ml). These results suggested that these effects were neither musculotropic nor competitive against ACh. In conclusion, CN-009 concentration-dependently inhibited the urethral contraction and the skeletal muscular twitch response. It was suggested that T-60 and GBX had different mechanisms and inhibited the response synergistically.

Animals↗

[Effects of cernitin pollen extract (CN-009) on the isolated bladder smooth muscles and the intravesical pressure].

Cernitin pollen extract (CN-009), extract from several pollen species, has been used for urinary dysfunction. As its mode of action has not been clarified, we investigated the action of CN-009 on the isolated bladder smooth muscles of rats, guinea pigs and cats and the intravesical pressure in female rats. CN-009 contracted isolated detrusor muscles of rats, guinea pigs and cats in a concentration-dependent manner. In the guinea pig detrusor muscle, the contractile effect of CN-009 was depressed by atropine, diphenhydramine and increased by cimetidine. In the rat detrusor muscle, the CN-009-induced contraction was depressed by atropine. In adult rats (11-23 weeks old) and aged rats (2 years old), CN-009 showed a dose-dependent increase of intravesical pressure to the same extent in spite of the fact that the aged rats had a lower responsiveness to acetylcholine. In adult rats, the CN-009-induced increase of intravesical pressure was reduced completely by atropine and partly reduced by phentolamine and guanethidine. Three weeks consecutive oral administration of CN-009 tended to increase the basal intravesical pressure and tended to elevate the isoproterenol-induced decrease and serotonin-induced increase in the intravesical pressure. These results suggest that CN-009 contracts the detrusor muscle, a process that is mainly mediated by muscarinic receptor activation. The contraction induced by CN-009 of detrusor muscle causes the increase of intravesical pressure.

Aging↗

A PCR-based marker for targeting small rye segments in wheat background.

We attempted to develop a PCR-based marker that detects various segments of rye chromosome incorporated into wheat. We designed three sets of PCR primers based on the nucleotide sequence data of a rye repetitive sequence previously reported. One of the primer sets amplified a clear ca. 1.4 kb fragment in a rye cultivar but not in any form of wheat, diploid, tetraploid or hexaploid. We used this critical primer set for PCR of various wild species and cultivars of rye, an array of wheat plants carrying different rye chromosomes or small segments from different regions of rye chromosome 1R, and plants carrying parts of the rye B chromosome. The PCR amplified the 1.4 kb fragment in all the plant materials examined. We believe this PCR primer set will be useful as a universal PCR-based marker for the introgression of rye chromosome segments in the wheat genome.

Chromosomes, Plant↗

Fluorescent labeling of plant chromosomes in suspension by FISH.

By optimizing the concentration and time of treatment with hydroxyurea (HU), a DNA synthesis inhibitor, and trifluralin, a microtubule inhibitor, a highly effective (over 60%) cell cycle synchronization method for rye and barley meristem cells was developed. Chromosome suspensions containing highly purified and morphologically intact rye and barley chromosomes were prepared from the meristems of their root tips by homogenization. Digoxigenin-labeled 5S rDNA was used as a probe in FISH for the rye chromosomes in the suspension, and biotin-labeled 17S rDNA and centromeric DNA were used in FISH for the rye and barley chromosome suspensions, respectively. Bright signals were detected at the specific regions of interest on the chromosomes. The results indicate that the method developed in this study is useful for selection and sorting of chromosomes that are not distinguishable by other means, using specific fluorescent labeling by FISH of the chromosomes in suspension.

Chromosomes, Plant↗

Limited proteolysis and reduction-carboxymethylation of rye seed chitinase-a: role of the chitin-binding domain in its chitinase action.

By a limited proteolysis with thermolysin, rye seed chitinase-a (RSC-a) was separated into a N-terminal cysteine-rich chitin-binding (CB-) domain (48 residues) and a catalytic (Cat-) domain (254 residues). The hydrolytic activity of the isolated Cat-domain toward soluble glycolchitin, was similar to that of RSC-a, but that toward insoluble colloidal chitin was 28% of that of RSC-a. Five disulfide bonds in the CB-domain were reduced with 2-mercaptoethanol (2-ME) in the absence of denaturing agents by an "all-or-none" process, that is, once the disulfide bond between Cys15 and Cys42 in the CB-domain was cleaved, the remaining four disulfide bonds were reduced very easily. The reduced and carboxymethylated RSC-a completely lost the chitin-binding ability, but retained 50% of the hydrolytic activity toward colloidal chitin of RSC-a. From these results, it was shown that RSC-a consists of a CB-domain and a Cat-domain connected by a flexible linker, and it was suggested that the CB-domain increases the hydrolytic action of Cat-domain toward insoluble chitin derivatives by binding to them.

Amino Acid Sequence↗

Identification of the aspartic acid residue located at or near substrate-binding site of rye seed chitinase-c.

Carboxyl groups of rye seed chitinase-c (RSC-c) were modified with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and glycine ethyl ester (GEE) at pH 5.5 and 5 degrees C in the presence and absence of (GlcNAc)4. In the absence of (GlcNAc)4, 5.2 carboxyl groups were modified by 90 min-reaction and the chitinase activity was reduced to 2.0%, while in the presence of (GlcNAc)4, 4.6 carboxyl groups were modified and 72% of the activity was retained. To identify the carboxyl group protected by (GlcNAc)4 from the modification, RSC-c was first modified with EDC and GEE in the presence of (GlcNAc)4 and then radiolabeled with EDC and [14C]GEE in the absence of (GlcNAc)4. Analyses of the radioactive peptides from the tryptic and chymotryptic digests of radiolabeled RSC-c showed that the main radiolabeled carboxyl group is that of Asp95, suggesting that Asp95 is located at or near substrate-binding site of RSC-c.

Acetylglucosamine↗

Phytotoxic sesterterpene, 11-epiterpestacin, from Bipolaris sorokiniana NSDR-011.

The structure of siccanol, a phytotoxic sesterterpene of fungal origin, was analyzed after chemical conversion by NMR spectroscopy. Siccanol was found to be an epimer of terpestacin that has been isolated from Arthrinium sp., and was thus renamed 11-epiterpestacin. Its stereochemistry was also identical with that of fusaproliferin, a structurally related mycotoxin from Fusarium proliferatum. Therefore, this sesterterpene may also be referred to as 24-deacetyl fusaproliferin. The phytotoxicity of 11-epiterpestacin was almost equal to that of terpestacin, but significantly higher than that of fusaproliferin.

Ascomycota↗

Alien DNA introgression and wheat DNA rearrangements in a stable wheat line derived from the early generation of distant hybridization.

Polyploidy has been found to be common in plants. Bread or common wheat (Triticum aestivum L., 2n=42) is a good example of allopolyploid made up of three diploid genomes A, B and D. In recent years, by the study of mimicking the origination of common wheat, it was found that changes of DNA sequence and gene expression occurred at the early stages of artificial allohexaploid between tetraploid wheat and Aegilops tauschii, which was probably favorable to genetic diploidization of new synthetic hexaploid wheat. Common wheat 99L2 is a new line stable in genetic, which was derived from the early self-pollinated generation of wide hybrids between common wheat and rye. In this study, it was found that at least two rye DNA segments had been introgressed into 99L2. This result suggested that a mechanism of alien DNA introgression may exist, which was different from the traditional mechanism of chromosome pairing and DNA recombination between wheat and alien species. Meanwhile, during the introgression process of alien rye DNA segments, the changes in DNA sequences of wheat itself occurred.

Base Sequence↗

[Transfer of a rye small chromosomal segment with powdery mildew-resistant gene(s) into common wheat (Triticum aestivum L.)].

The advanced progeny lines (BC1F5) from the monosomic addition lines between common wheat cultivar Mianyang 11, which is highly susceptible to powdery mildew, and an inbred rye line R12 were analyzed for selection of wheat-rye translocations. Based on a rye-specific repetitive sequence of pSc20H, which spread over all chromosomes of rye but did not existed in wheat, a set of PCR primer was designed and used to identify the rye chromosome segments in wheat. From 300 of the BC1F5 progeny lines 70 were found to contain chromosome composition of rye. An advanced line, 96II691-830-98, originated from 6R monosomic addition line was observed to be immune to powdery mildew, different from its wheat parent Mianyang 11. A small segment of rye chromosome at telomere in a pair of wheat chromosome in the line was found by means of GISH. The results indicated that a small segment of rye chromosome 6R carrying the gene(s) for resistance to powdery mildew has been transferred into common wheat. In the progeny of monosomic addition lines a high frequency of wheat-alien species translocation with various segments of chromosomes could be found by application of both PCR and GISH technique.

Chromatin↗

Haemolytic properties of cereal 5-n-alk(en)ylresorcinols.

The haemolytic activity of 5-n-alk(en)ylresorcinols is temperature dependent and correlated to their transition temperatures. The values of the parameters describing the 5-n-alk(en)ylresorcinol-induced red blood cell lysis indicate strong affinity of the compounds to the membrane and their high lytic capacity. The affinity of the compounds for the membrane decrease with the increasing quantity of the molecules incorporated into the erythrocyte membrane and is much higher for saturated resorcinols than unsaturated ones. The amount of 5-n-alk(en)ylresorcinol molecules bound to the membrane at a hundred percent lysis is about eighty times and eleven times (for alkyl and alkenyl derivatives respectively) higher than at zero percent lysis. Estimated free energy of erythrocyte lysis was similar for alkyl and alkenyl derivatives of resorcinol provided the preparation of the resorcinolic suspensions above their transition temperatures.

Edible Grain↗