The role of calcium ions in phytochrome-controlled swelling of etiolated wheat (Triticum aestivum L.) protoplasts

Protoplasts from dark-grown wheat (Triticum aestivum L.) maintained at a constant osmotic potential at 22°C, were found to swell upon red irradiation (R) and the effect was negated by subsequent far-red light (FR), indicating phytochrome involvement. Swelling only occurred when Ca²⁺ ions were present in the surrounding medium, or were added within 10 min after R. Furthermore, Mg²⁺, Ba²⁺ or K⁺ could not replace this requirement for Ca²⁺. The presence of K⁺ did not enhance the Ca²⁺-dependent swelling response. When the Ca²⁺-ionophore A 23187 was added to the medium, protoplasts swelled in the dark to the same extent as after R. Both the Ca²⁺-channelblocker Verapamil and La³⁺ inhibited R-induced swelling. It is proposed that R causes the opening of Ca²⁺-channels in the plasma membrane. Boyle-van't Hoff analyses of protoplast volume after R and FR are consistent with the conclusion that R irradiation causes changes in membrane properties.Abbreviations EDTA ethylenediaminetetraacetic acid - FR far-red light - nov non-osmotic-volume - Pfr FR-absorbing form of phytochrome - Pr R-absorbing form of phytochrome - R red light     

Barley γ3-hordein: Glycosylation at an atypical site,disulfide bridge analysis,and reactivity with IgE from patients allergic to wheat

Post translational modifications of a seed storage protein, barley γ3-hordein, were determined using immunochemical and mass spectrometry methods. IgE reactivity towards this protein was measured using sera from patients diagnosed with allergies to wheat. N-glycosylation was found at an atypical Asn-Leu-Cys site. The observed glycan contains xylose. This indicates that at least some γ3-hordein molecules trafficked through the Golgi apparatus. Disulfide bridges in native γ3-hordein were almost the same as those found in wheat γ46-gliadin, except the bridge involving the cysteine included in the glycosylation site. IgE reacted more strongly towards the recombinant than the natural γ3-hordein protein. IgE binding to γ3-hordein increased when the protein sample was reduced. Glycosylation and disulfide bridges therefore decrease epitope accessibility. Thus the IgE from patients sensitized to wheat cross-react with γ3-hordein due to sequence homology with wheat allergens rather than through shared carbohydrate determinants.     

Modified culture conditions for increased viability and cell wall synthesis in grapevine (Vitis vinifera L. cv. Sultanina) leaf protoplasts

Studies were undertaken to determine optimum conditions for grapevine protoplast culture. Highest viability was obtained at 25° in the dark, and at initial pH values of 5.2 to 7.2, in the presence of 1 or 2% (w/v) sucrose and 0.6 or 0.7 M mannitol or osmolality between 729 and 930 mOsmol kg^-1. Optimum plant growth regulators were 6-BAP/NAA at 2.3×10^-6/10–15×10^-6 M, respectively. ³⁵S-Methionine incorporation into de novo synthesized proteins for protoplasts of Nicotiana tabacum cv Xanthi (a readily regenerating species) and for the recalcitrant grapevine was studied. In tobacco the rate of protein synthesis showed 3 maxima which coincided with the distinct stages of naked protoplasts, cell wall reconstitution and induction of cell division. In grapevine protoplasts the first peak exceeded the second one, whereas no peak corresponding to the induction of cell division was observed.     

Chromosomal location of a gene for resistance to septoria tritici blotch (Mycosphaerella graminicola)in the hexaploid wheat ’Synthetic 6x’

Septoria tritici blotch, caused by the fungus Mycosphaerella graminicola,is currently the major foliar disease of wheat world-wide, and new sources of resistance and knowledge about the genetics of resistance are needed to improve breeding for resistance to this disease. Sears’s ’Synthetic 6x’ hexaploid wheat, derived from a hybrid of Triticum dicoccoides and Triticum tauschii, was resistant to 12 of 13 isolates of M. graminicola tested. Chromosome 7D of ’Synthetic 6x’ was identified as carrying resistance to all 12 isolates in tests of seedlings of inter-varietal chromosome substitution lines of ’Synthetic 6x’ into ’Chinese Spring’ and to two isolates in tests of adult plants. A septoria tritici blotch resistance gene, named Stb5, was identified using the M. graminicola isolate IPO94269 and mapped on the short arm of chromosome 7D, near the centromere, in a population of single homozygous chromosome-recombinant lines for the 7D chromosome. Received: 1 February 2001 / Accepted: 17 April 2001     

Seasonal and Diel Changes in Behaviour,Microhabitat use and Preferences by Young Pool-dwelling Atlantic Salmon,Salmo salar,and Brown Trout,Salmo trutta

There was a pronounced decline in activity of young pool-dwelling Atlantic salmon, Salmo salar, and brown trout, Salmo trutta, as the water temperatures dropped in the autumn and early winter, and the fish switched from a predominantly diurnal towards a nocturnal activity pattern. Such a switch in activity pattern has previously been observed in young brown trout, but the present study is the first documentation for juvenile Atlantic salmon under natural conditions. Juvenile fish fed actively even when water temperatures were below 0°C, although foraging behaviour at near-freezing temperatures was recorded exclusively during night surveys. This indicates that other proximate factors, in addition to water temperature, affect the activity of young salmon and trout in rivers. Trout kept feeding positions significantly higher above bottom than salmon in August and September, but both species reduced the height above bottom at the onset of winter, possibly due to reduced swimming performance and lowered food availability in the upper part of the water column.     

The effect of external Ca^2＋ and Ca^2＋—channel modulators on red—light—induced swelling of protoplasts of Phaseolus radiatus L.

Red-light-induced swelling of the protoplasts isolated from hypocotyl of etiolated mung bean(Phaseolus radiatusL.)was observed only when Ca^2 ions were present in the medium.The optimal CaCl2 concentration was 250μM,Swlling response declined when Ca^2 was supplied into the medium after red light irradiation.The Ca^2 -chelator EGTA eliminated the red-light-induced swelling and 45Ca^2 accumulation in the protoplasts.In conltrast,A23187,a Ca^2 -ionophore,could mimic the effect of red light in darkness.These results indicate that Ca^2 may play a role in light signal transduction.In addition,swelling response was prevented by TFP and CPZ(both are CaM antagonists),implying the involvement of CaM in red-light-induced and Ca^2 -dependent protoplast swelling.     

Chlorophyll b accumulation and grana formation in low intensities of red light

Abstract When dark grown leaves of wheat (Triticum aesivum L.) were given a brief irradiation, there was an immediate onset of chlorophyll (Chl) b synthesis in the dark. This synthesis led to a rather slow accumulation of Chl b, which ceased when the Chl b/Chl a ratio had reached a value of about 0.1. The Chl b synthesis occurred also when the seedlings were treated with the herbicide SAN 9789. Leaves grown under different intensities of red light accumulated Chl b and Chl a, resulting in a ratio Chl b/Chl a which depended on the light intensity. If the light intensity was low, Chl a accumulated to a level about ten times the level of PChlide of the dark grown leaves. This occurred without any increase in the Chl b/Chl a ratio. There was no difference between SAN 9789-treated seedlings and water controls in this respect. Above a certain threshold of irradiance, the Chl b/Chl a ratio in the control leaves increased rapidly with the irradiation intensity. The increase in Chl b/Chl a ratio coincided with formation of grana in the plastids. This increase was not found and grana formation was completely absent in the seedlings treated with SAN 9789. The possibility of two different stages in the Chl b synthesis is discussed.     

Production of male sterile interspecific somatic hybrids between transgenic N. tabacum (Bar) and N. rotundifolia (NPT II) and their identification by aflp analysis

Summary A protoplast fusion experiment was carried out aiming to obtain somatic hybrid plants of transgenic Nicotiana tabacum (bar) (+) N. rotundifolia (npt II). The bialaphos resistance marker (bar) was introduced into N. tabacum via Agrobacterium tumefaciens using vector pGV1500 carrying the bar gene phosphinothricin acetyltransferase. N. rotundifolia (npt II) was recovered after direct gene transformation of protoplasts by the pGP6 plasmid carrying the npt II gene for neomycin phosphotransferase. Both plasmids possessed 35S CaMV promotors. Hybrid selection was based on dual bialaphos— kanamycin resistance. Amplified fragment length polymorphism (AFLP) analysis of regenerated plants showed the presence of species-specific bands for both parents, which confirmed their hybrid nature. N. tabacum (bar) (+) N. rotundifolia (npt II) hybrids exhibited a great diversity in morphology. Fertile hybrids which possessed N. tabacum or N. rotundifolia morphology were recovered. Flow cytometric analysis revealed that the N. tabacum- and N. rotundifolio-like hybrids had nuclear DNA contents near that of N. tabacum (9.40±0.24pg) or N. rotundifolia (5.29±0.36 pg), respectively, and were highly asymmetric. Other hybrids combined traits from the two species at various levels—N. tabacum habit or branched, similar to N. rotundifolia. Their leaves varied in shape. The flowers of the hybrid plants were of N. tabacum or N. rotundifolia type, or had N. rotundifolia dimensions, pink with N. tabacum corolla or white with curly fused petals. All were self-sterile or male sterile. The nuclear DNA content varied from 8.90±0.30 to 19.57±0.33 pg. The data from the morphological and cytological analysis provided vidence that parental chromosome elimination in the hybrid clones was spontaneous and not species-specific and that diploidization of the tobacco genome might have occurred in some clones during in vitro culture. This reflects the genomic incompatibility between the two species.     

Biochemical and molecular identification of Xanthomonas translucens pv. undulosa causing bacterial leaf streak of wheat in Punjab,Pakistan

Xanthomonas translucens pv. undulosa, (Xtu.), causal agent of Bacterial leaf streak (BLS) of wheat, was characterised through pathogencity, hypersensitivity, biochemical and molecular assays. Fifty symptomatic leaves of wheat were collected from eight agro-ecological zones of Punjab out of which 25 were isolated and purified. Maximum incidence and severity in Faisalabad were followed by Multan and Rahim Yar Khan. The pathogen isolated from diseased leaves was identified on the basis of colonies pattern, colour, biochemical and pathogencity test as X. translucens pv. undulosa and confirmed its pathogencity through pathogencity test. For molecular characterization, the bacterial 16S–23S rDNA spacer fragments were amplified by PCR with conserved primers (C1 and C2) and then in combination with specific primers (T1 & T2). 300?bp product amplified by C1 and C2 primer pair confirmed the presence of Xanthomonas, while specific primers T1 and T2 amplified a product of 200?bp, confirmed the presence of X. translucens pv. undulosa. This work will be quite helpful for wheat pathologist and breeders for future management strategy for this disease.