Resistance to recombinant stem rust race TPPKC in hard red spring wheat.

The wheat (Triticum aestivum L.) stem rust (Puccinia graminis Pers.:Pers. f.sp. tritici Eriks. and Henn.) resistance gene SrWld1 conditions resistance to all North American stem rust races and is an important gene in hard red spring (HRS) wheat cultivars. A sexually recombined race having virulence to SrWld1 was isolated in the 1980s. Our objective was to determine the genetics of resistance to the race. The recombinant race was tested with the set of stem rust differentials and with a set of 36 HRS and 6 durum cultivars. Chromosomal location studies in cultivars Len, Coteau, and Stoa were completed using aneuploid analysis, molecular markers, and allelism tests. Stem rust differential tests coded the race as TPPKC, indicating it differed from TPMKC by having added virulence on Sr30 as well as SrWld1. Genes effective against TPPKC were Sr6, Sr9a, Sr9b, Sr13, Sr24, Sr31, and Sr38. Genetic studies of resistance to TPPKC indicated that Len, Coteau, and Stoa likely carried Sr9b, that Coteau and Stoa carried Sr6, and Stoa carried Sr24. Tests of HRS and durum cultivars indicated that five HRS and one durum cultivar were susceptible to TPPKC. Susceptible HRS cultivars were postulated to have SrWld1 as their major stem rust resistance gene. Divide, the susceptible durum cultivar, was postulated to lack Sr13. We concluded that although TPPKC does not constitute a threat similar to TTKSK and its variants, some cultivars would be lost from production if TPPKC became established in the field.

High temporal resolution in situ measurement of the effective particle size characteristics of fluvial suspended sediment.

This paper reports the use of a LISST-100 device to monitor the effective particle size characteristics of suspended sediment in situ, and at a quasi-continuous temporal resolution. The study site was located on the River Exe at Thorverton, Devon, UK. This device has not previously been utilized in studies of fluvial suspended sediment at the storm event scale, and existing studies of suspended sediment dynamics have not involved such a high temporal resolution for extended periods. An evaluation of the field performance of the instrument is presented, with respect to innovative data collection and analysis techniques. It was found that trends in the effective particle size distribution (EPSD) and degree of flocculation of suspended sediment at the study site were highly complex, and showed significant short-term variability that has not previously been documented in the fluvial environment. The collection of detailed records of EPSD facilitated interpretation of the dynamic evolution of the size characteristics of suspended sediment, in relation to its likely source and delivery and flocculation mechanisms. The influence of measurement frequency is considered in terms of its implications for future studies of the particle size of fluvial suspended sediment employing in situ data acquisition.

Ligand-receptor dissociation: a potential mechanism for the attenuation of estrogen action in the juvenile rabbit uterus.

Estradiol binding kinetics and receptor activation were investigated using cytosol estrogen receptor from adult rabbit uterine endometrium and from the undifferentiated uteri of 2-week-old rabbits. The cytosol estrogen receptor from juvenile compared to that from adult rabbit uteri was lower (P less than 0.01) in concentration, was associated with reduced (P less than 0.01) titers of serum estradiol, and had a lower affinity for estradiol (Ka = 10(7) M-1). The equilibrium association constant (Ka) for the estrogen receptor from juvenile uteri was reduced by an increase in the dissociation rate constant (kd), as measured by [3H]E2 dissociation from the receptor. Enhanced steroid-receptor dissociation in juvenile uteri was correlated with a reduced rate (P less than 0.01) of receptor activation, as measured by the binding of steroid-receptor complex to DNA-cellulose. Because receptor activation was limited at elevated temperature (30 C), activation studies were performed at low temperature (0 C), and under these optimum conditions, the change in binding kinetics observed in the juvenile was correlated with a reduced rate of receptor activation. Equilibrium binding of [3H]E2 to the estrogen receptor exhibited positive cooperativity, as indicated by Hill coefficients of 3.39 +/- 0.12 and 3.44 +/- 0.11 for juveniles and adults, respectively. The ratio of bound to free steroid was decreased in cytosol from juvenile compared to adult uteri. Collectively, these results support the hypothesis that the increased off rate and decreased activation rate of estrogen receptor in immature rabbit uteri may represent a mechanism for the attenuation of estrogen action before sexual maturation.

Injury and repair in biocide-treated spores of Bacillus subtilis.

Bacillus subtilis NCTC 8236 spores exposed to appropriate concentrations of test biocides (glutaraldehyde, two iodine and two chlorine preparations) were able to repair injury if subsequently held in nutrient broth at 37 degrees C but not in broth at 22 degrees C, sterile filtered water at 4, 22 or 37 degrees C or germination medium at 37 degrees C. Repair appeared to occur primarily during outgrowth and was initiated soon for iodine-treated spores and latest for glutaraldehyde-treated ones.

The nature and site of biocide-induced sublethal injury in Bacillus subtilis spores.

Spores of Bacillus subtilis NCTC 8236 exposed at 22 degrees C to test biocides (alkaline glutaraldehyde, an iodophor, Lugol's solution, sodium hypochlorite and sodium dichloroisocyanurate) demonstrated varying degrees of injury to stressing agents (sodium hydroxide, sodium lauryl sulphate, polymyxin B sulphate or cetylpyridinium chloride) incorporated into a recovery agar medium. This injury to stressing agents was expressed mainly during outgrowth.

Revival of biocide-treated spores of Bacillus subtilis.

Spores of Bacillus subtilis NCTC 8236 were treated with biocides and then subjected to various revival procedures. Sodium hydroxide (optimum concentration 25 mmol l-1) revived a small portion of glutaraldehyde-treated spores but not of spores exposed to formaldehyde, polyvinylpyrrolidone-iodine (PVP-I), Lugol's iodine, sodium hypochlorite or sodium dichloroisocyanurate (NaDCC). Post-treatment heat shock (at 70 degrees or 80 degrees C) increased the numbers of colony-forming units (cfu) of formaldehyde-injured spores. Coat-extraction procedures had the greatest effect on iodine-pretreated spores. The uptake of iodine and chlorine was more rapid and occurred to a greater extent with outgrowing, germinating and especially coat-deficient spores than with mature, resting spores.

The effects of some halogen-containing compounds on Bacillus subtilis endospores.

Sodium hypochlorite (NaOCl) and sodium dichloroisocyanurate (NaDCC) were more active against Bacillus subtilis 8236 spores in both viability and in germination and outgrowth studies than were polyvinylpyrrolidone-iodine (PVP-I) and Lugol's solution. Of the two chlorine compounds studied NaOCl proved to be the more active. The two iodine-containing compounds gave contrasting results with the Lugol's solution demonstrating increased antibacterial activity with increasing available iodine concentration. The antibacterial behaviour of PVP-I, however, reflected the more complex nature of aqueous iodine-surfactant mixtures. Initially, non-complexed iodine concentration (the active species) increased with increasing total available iodine concentration, resulting in increasing antibacterial activity. However, due to changes in the physical properties of the mixture, a maximum concentration of non-complexed iodine was reached so that a further increase in total available iodine resulted in a decrease in non-complexed iodine concentration and consequently a decrease in the antibacterial activity of the solution was observed. A greater inhibitory effect was observed in subsequent germination and outgrowth studies when spores were pre-treated with respective biocide than when untreated spores were added to germination media containing biocide at t = 0. This may reflect a combination of different contact times plus the neutralizing effect of the germination media on such halogen compounds.

Revival of Bacillus subtilis spores from biocide-induced injury in the germination process.

Spores of Bacillus subtilis NCTC 8236 were treated with glutaraldehyde, Lugol's iodine, polyvinylpyrrolidone-iodine (PVP-I), sodium hypochlorite or sodium dichloroisocyanurate (NaDCC). After exposure survivors were enumerated on nutrient agar containing potential revival agents (subtilisin, lysozyme, calcium dipicolinate, calcium lactate). Of these, only calcium lactate had any significant enhancing effect and then only with iodine-treated spores. Calcium lactate (9 mmol l-1) in nutrient broth enhanced the rate and extent of germination of iodine-treated spores but not of spores previously subjected to glutaraldehyde, hypochlorite or NaDCC.

Conditions suitable for the recovery of biocide-treated spores of Bacillus subtilis.

Various factors were studied in order to determine the optimum conditions for the recovery of Bacillus subtilis spores treated with two iodine preparations, two chlorine-releasing agents or glutaraldehyde. The composition of the recovery medium was not usually important except that counts on brain heart infusion agar were significantly lower than on other media for iodine-treated spores. The addition to recovery media of soluble starch, charcoal, D-glucose or yeast extract usually had no discernible beneficial effect on colony counts. Maximum counts of survivors were obtained after an incubation period of 3 days and an optimum incubation temperature of 30 or 37 degrees C. Germination and outgrowth of biocide-exposed spores were more sensitive to changes in incubation temperature than were control spores.

Characterization of a mitotic mutant of durum wheat.

An ethyl methanesulfonate-induced mitotic mutant of durum wheat (Triticum turgidum L. var. durum; 2n = 4x = 28) was found. We have characterized the mutant to determine the mechanism of abnormal cell division and to test for temperature effects on abnormal cell division. Stained root-tip meristems and pollen mother cells were studied with brightfield, phase contrast, and immunofluorescence microscopy. Abnormal cells included metaphase cells with a multiple of the normal complement (8x = 56, or 16x = 112), multinucleate cells, 4C, 8C, or 16C mononucleate cells, and cells exhibiting incomplete cytokinesis. The mutant had three classes of pollen mother cells: euploid with normal bivalent pairing, multiploid with bivalent pairing, and multiploid with multivalent pairing. Preprophase bands and spindles were normal in mononucleate cells. Some cells had asymmetrical phragmoplasts and phragmoplast dismantling that produced incomplete cytokinesis. Failure of cytokinesis followed by nuclear fusion were the mechanisms of abnormal cell division. To test for temperature sensitivity of the mutant, seedlings were germinated under six different temperature regimes. As germination temperature increased, the frequency of abnormal cells increased. When the mutant was crossed as the female with durum wheat, 3% of hybrids were hexaploid, indicating that functional-unreduced gametes had formed in megaspores.

Wheat lines monogenic for resistance to stem rust from the wheat cultivar 'Waldron'.

The Triticum aestivum L. cultivar 'Waldron' has long lasting resistance to most North American stem rust (Puccinia graminis Pers.:Pers. f. sp. tritici Eriks. and E. Henn.) isolates. The objective of this research was to develop wheat lines monogenic for resistance to stem rust from 'Waldron' using allelism tests and tests for reaction to a series of ten stem rust cultures having a range of virulences. Twelve lines homozygous for single resistance genes were selected as parents of a diallel cross to test for allelism among genes for resistance. We identified 6 lines or groups of lines (WDR-A1, the WDR-B1 and WDR-B2 group, the WDR-C1 and WDR-C2 group, WDR-D1, the WDR-E1, WDR-E2, WDR-E3, and WDR-E4 group, and WDR-F1) that carried different single genes for resistance from 'Waldron'. A seventh line (WDR-G1) probably has two genes for resistance, one in common with WDR-C1 and WDR-C2. The gene in the WDR-E group is probably the same as SrWld1, and the one in WDR-F1 the same as Sri11. 'Waldron' probably has two or more genes for resistance to stem rust that previous genetic studies did not detect.

Chromosomal location of genes for gliadin polypeptides in durum wheat Triticum turgidum L.

The chromosomal location of genes was determined for 19 of 30 gliadin bands extracted from seeds of a set of durum wheat (Triticum turgidum L. var. 'durum') aneuploids and durum cultivars. Individual bands were identified by their relative mobility on polyacrylamide gels. The gene(s) for gliadin band 45, which has been associated with strong gluten by several authors, was shown to be controlled by chromosome 1 B. A band with similar mobility (band 46) was controlled by 'Chinese Spring' chromosome 1D. Conventional breeding procedures coupled with the use of electrophoresis of gliadin polypeptides should result in rapid conversion of current weaker gluten durum cultivars to strong gluten cultivars.

Chromosomal location of genes for stem rust resistance derived from 'Waldron' wheat.

The chromosomal locations of genes for resistance to stem rust (Puccinia graminis Pers.: Pers. f. sp. tritici Eriks. & E. Henn.) in the wheat (Triticum aestivum L.) cultivar 'Waldron' (WDR) were determined by monosomic analyses. Wheat lines WDR-B1, -C2, -E4, and -F1,which have single genes for resistance to stem rust derived previously from WDR sel. 'Little Club', were crossed onto a complete set of 21 'Chinese Spring' monosomics. The F2 and backcross-F1 (BC1F1) seedlings from each of the 84 crosses were tested for reaction to culture 111-SS2 (CRL-LCBB) of stem rust, and a few selected segregants were analyzed cytologically for chromosome number. The F2 from 2 crosses of WDR-C2, -E4 and -F1 and the BC1F1 from 2 crosses of WDR-F1 were tested also with culture Or11c (CRL-QBCN). Significant deviations from disomic ratios towards monosomic ratios in the F2 and BC1F1 were used to determine which chromosomes carried the genes for resistance. Cytological analyses of certain BC1F1 and susceptible F2 plants were used to help identify the location of the genes for rust resistance. WDR-B1 has a gene, herein designated Sr41, for resistance on chromosome 4D. WDR-C2 has a gene on chromosome 7 A that may be the same as one previously designated SrWld2. WDR-E4 has a gene on chromosome 2A, possibly SrWld1, which is effective against most or all North American stem rust cultures. WDR-F1 has a gene on chromosome 6B that is the same as or similar to Sr11.

Electron transport and chloroplast ultrastructure of a chlorophyll deficient mutant of wheat.

A non-lethal chlorophyll deficient mutation was induced by use of the chemical mutagen ethyl methanesulfonate. Chloroplasts from the control and mutant plants were found to be very similar ultrastructurally. Thylakoid membrane volume was only slightly greater in plastids from the control as compared with plastids from the mutant. The chlorophyll content of the mutant was reduced by over 60%. This decrease in chlorophyll was not accompanied by a similar decrease in electron transport. Uncoupled electron transport rate based on a unit chlorophyll basis was nearly twice as great for mutant chloroplasts as for control plastids. However, electron transport rate based on a unit membrane volume was similar in mutant and control plants. At high irradiance the relative quantum requirement of the control and mutant was similar when expressed on membrane volume.

Regulation of excitation energy in a wheat mutant deficient in light-harvesting pigment protein complex.

Chloroplasts of the CD3 wheat mutant were deficient primarily in chlorophyll of light harvesting pigment proteins (LHPP) 1 and 2 and CP1a. The reduced level of protein associated with chlorophyll of LHPP1 and LHPP2 and the reduced level of low molecular weight polypeptides between 23 and 29 kilodaltons confirmed that the CD3 mutant was deficient in the LHPP complex. The high fluorescence emission ratio at 740 (F740) to 686 nanometers (F686) observed from chloroplasts of normal wheat following light induced phosphorylation of the LHPP complex was not noted from mutant chloroplasts. The long wavelength peak fluorescence emission (F740) was shifted to a shorter wavelength peak (F725) and was reduced in intensity compared to that of normal wheat thylakoids. The ratio of variable fluorescence to maximum fluorescence, a measure of PSII photochemical efficiency, was the same for the normal wheat and mutant leaves. The ratios of uncoupled photosystem I/photosystem II electron transport rates for mutant and normal wheat chloroplasts were similar at saturating light suggesting that absorbed excitation energy was distributed to the two photosystem reaction centers of the mutant in a similar manner as in the normal wheat. Proteins of the LHPP complex were differentially phosphorylated by action of a membrane protein kinase when both normal wheat and CD3 mutant thylakoids were irradiated without an electron transport chain acceptor. Even though the F740/F686 ratio was low in mutant thylakoids, the phosphorylation of the 27-kilodalton LHPP polypeptide was consistent with the mutant being in a state II condition. The data gave rise to the suggestion that the F740/F686 ratio might not indicate excitation energy distribution to the two photosystems in the mutant.

Acidic lipids enhance cathepsin D cleavage of the myelin basic protein.

Some acidic lipids including sulfatide and phosphatidylinositol were found to increase greatly the rate of cathepsin D cleavage of the myelin basic protein. Since a similar effect was seen when the substrate was changed to cytochrome C, but not when the enzyme was changed to pepsin, these acidic lipids seem to be acting on cathepsin D rather than on myelin basic protein itself. Even so, chemical modification studies suggest that this phenomenon is only seen when the myelin basic protein is in its native conformation. Succinylation of MBP increases its rate of cleavage by cathepsin D by at least tenfold and, in addition, with this modified and presumably denatured MBP as substrate, activation of cathepsin D is no longer seen with acidic lipids. These findings suggest that the native conformation of MBP is both an important determinant of its rate of cleavage by cathepsin D and is also essential for observing activation of this reaction by acidic lipids. The acidic lipids seem to alter the "extended active site" of cathepsin D in such a way as to enable this enzyme better to utilize the native myelin basic protein as a substrate. Cathepsin D has previously been implicated as the protease responsible for the release into cerebrospinal fluid in multiple sclerosis patients of an encephalitogenic fragment derived from myelin basic protein. It is possible that the elevated levels of cathepsin D and sulfatide that have previously been found associated with multiple sclerosis plaques in vivo act in concert to bring about the rapid cleavage and subsequent loss of the myelin basic protein from these localized regions in the myelin sheath.

Chloramphenicol stimulation of light harvesting chlorophyll protein complex accumulation in a chlorophyll B deficient wheat mutant.

As compared with normal wheat leaves, the chlorina wheat mutant, designated CD3, has a high chlorophyll a/b ratio and a deficiency in the light harvesting chlorophyll protein (LHCP) complex. Applications of 200 micrograms per milliliter of d-threo-chloramphenicol to etiolated seedlings decreased the chlorophyll a/b ratio and increased the accumulation of the 27 kilodalton LHCP polypeptide and the LHCP complex in thylakoids of the mutant during greening. These data led to the suggestion that a protein encoded in chloroplast genes impaired either transcriptional, translational, or posttranslational events in CD3 wheat limiting the accumulation of the LHCP complex. The LHCP complex which accumulated in chloramphenicol treated wheat appeared functional even though chlorophyll protein complex accumulations were altered greatly in the wheat thylakoids. LHCP polypeptides were phosphorylated by action of a membrane protein kinase but yet photosystem II electron transport was impaired. The chloramphenicol treatment increased the photosystem I/photosystem II ratio of electron transport and the fluorescence emission ratio at 740 to 686 nanometers relative to those of untreated wheat. Chloramphenicol prevented development of normal granal thylakoids in normal wheat chloroplasts but not in those of the CD3 mutant. Elongated stacked thylakoids were observed in normal wheat. Net-like membranes and vesicles were noted in the stroma of chloroplasts from treated mutant seedlings.