K+(Rb+) and Ca2+ fluxes in young winter wheat plants exposed to sub-zero temperatures

Ice crystal formation temperature was determined in the region of the crown in one group of 7-day-old intact unhardened high-salt plants of winter wheat (Triticum aestivum L. cv. Weibulls Starke II) with TA (Thermal Analysis) and DTA (Differential Thermal Analysis) methods. After exposure of another group of plants, grown for the first 7 days in the same way as the first group, to various sub-zero temperatures (-1 to 5°C), influx in roots of Rb⁺(⁸⁶Rb⁺) and Ca²⁺(⁴⁵Ca²⁺) and contents of K⁺ and Ca²⁺ were determined at intervals during 7 days of recovery. Ice crystal formation in the crown tissue was probably extracellular and took place at about -4°C. There was a large loss of K⁺ from the roots after treatment at sub-zero temperatures. This loss increased as the temperature of the sub-zero treatment decreased. During recovery, roots of plants exposed to -1, -2 and -3°C gradually reabsorbed K⁺. Reabsorption of K⁺ in roots of plants exposed to -4°C was greatly impaired. Rb⁺ influx decreased and Ca²⁺ influx increased after sub-zero temperature treatments of the plants. Active Rb⁺ influx mechanisms and active extrusion of Ca²⁺ were impaired or irreversibly damaged by the exposure. While Rb⁺ influx mechanisms were apparently repaired during recovery in plants exposed to temperatures down to -3°C, Ca²⁺ extrusion mechanisms were not. The temperature for ice crystal formation in the region of the crown tissue coincides with the temperature at which the plants lost the ability to reabsorb K⁺ and to repair Rb⁺ influx mechanisms during the recovery period. Plants were lethally damaged at temperatures below ?4°C.     

Variability and genetics of spacer DNA sequences between the ribosomal-RNA genes of hexaploid wheat (Triticum aestivum)

Summary Using restriction enzyme digests of genomic DNA extracted from the leaves of 25 hexaploid wheat (Triticum aestivum L. em. Thell.) cultivars and their hybrids, restriction fragment length polymorphisms of the spacer DNA which separates the ribosomal-RNA genes have been examined. (From one to three thousand of these genes are borne on chromosomes 1B and 6B of hexaploid wheat). The data show that there are three distinct alleles of the 1B locus, designated Nor-B1a, Nor-B1b, and Nor-B1c, and at least five allelic variants of the 6B locus, designated Nor-B2a, Nor-B2b, Nor-B2c, Nor-B2d, and Nor-B2e. A further, previously reported allele on 6B has been named Nor-B2f. Chromosome 5D has only one allelic variant, Nor-D3. Whereas the major spacer variants of the 1B alleles apparently differ by the loss or gain of one or two of the 133 bp sub-repeat units within the spacer DNA, the 6B allelic variants show major differences in their compositions and lengths. This may be related to the greater number of rDNA repeat units at this locus. The practical implications of these differences and their application to wheat breeding are discussed.     

Regeneration of Triticum aestivum apical explants after microinjection of germ line progenitor cells with DNA

A highly efficient method of regenerating fertile, phenotypically normal plants from shoot apex cultures of T. aestivum was developed. The hypodermal layer (L2) of the vegetative apex containing germ line precursor cells could be located with bright field microscopy and targeted for microinjection. Fluorescently labelled dextrans were used as markers to develop a microinjection procedure which did not disrupt nuclear or cytoplasmic structure. This procedure was used to inject plasmid DNA into L2 cells. Capillary microinjection did not shear the plasmid DNA and delivery of DNA was confirmed by polymerase chain reaction analysis of DNA isolated from injected apices. The significance of these findings in relation to the development of cereal transformation systems will be discussed.     

5S ribosomal gene clusters in wheat: pulsed field gel electrophoresis reveals a high degree of polymorphism

Summary The long-range structure of 5S rRNA gene clusters has been investigated in wheat (Triticum aestivum L.) by means of pulsed field gel electrophoresis. Using aneuploid stocks, 5S rRNA gene clusters were assigned to sites on chromosomes 1B, 1D, 513 and 5D. Cluster sizes were evaluated and the copy number of 5S DNA repeats was estimated at 4700-5200 copies for the short repeating unit (410 bp) and about 3100 copies for the long repeat (500 bp) per haploid genome. A comparison of wheat cultivars revealed extremely high levels of polymorphism in the 5S rRNA gene clusters. With one restriction enzyme digest all varieties tested gave unique banding patterns and, on a per fragment basis, 21-fold more polymorphism was detected among cultivars for 5S DNA compared to standard restriction fragment length polymorphisms (RFLPs) detected with single copy clones. Experiments with aneuploid stocks suggest that the 5S rRNA gene clusters at several chromosomal sites contribute to this polymorphism. A number of previous reports have shown that wheat cultivars are not easily distinguished by isozymes or RFLPs. The high level of variation detected in 5S rRNA gene clusters therefore offers the possibility of a sensitive fingerprinting method for wheat. 5S DNA and other macro-satellite sequences may also serve as hypervariable Mendelian markers for genetic and breeding experiments in wheat.     

Changes in Wheat Leaf Extracellular Proteolytic Activity after Infection with Septoria tritici

The proteolytic activity of the leaf extracellular space of wheat cultivars Pigüé and Isla Verde was estimated after inoculation of either detached leaves or plants with the fungus Septoria tritici. Pigüé is resistant, whereas Isla Verde is susceptible to the disease caused by S. tritici. Viable conidiospores of the fungus caused similar increases in both hydrogen peroxide production and chitinase activity of the cultivars studied. In contrast, they caused a decrease in the extracellular serine proteinase activity of Isla Verde and a significant increase in that of Pigüé. Independently of the cultivar from which it was extracted, the extracellular serine proteinase inhibited the germination of Septoria tritici conidiospores. These results suggest that the proteolytic activity of the leaf extracellular space can participate in the defence of wheat plants against Septoria tritici. Its regulation may be controlled by specific defence components of each cultivar.     

Genetically controlled differences in the effects of chlormequat on tetraploid wheat (Triticum turgidum)

Two crosses between Triticum turgidum wheat lines differing in their response to chlormequat (CCC) were tested. In the F₂ population of one cross, which was segregating for the Rht1 dwarfing allele, each plant was cloned by separation of two tillers, one of which was treated with CCC. The tall (rht1/rht1) and the intermediate (Rht1/rht1) genotypes showed a greater response to CCC than the semi-dwarf (Rht1/Rht1) genotype, as expressed by culm length and date of ear emergence. The F₃ families of another cross and their two semi-dwarf parents were grown in a three-replicated field test in paris of rows, one of which was treated with CCC. In one of the parents and in 1/4 of the F₃ families CCC induced a wide-angled tiller growth, suggesting a monogenic control of this growth habit in response to CCC.Based on an M.Sc. thesis presented by the senior author to the Faculty of Agriculture of The Hebrew University of Jerusalem.     

Mg2+-ATPase activity in wheat root plasma membrane vesicles: Time-dependence and effect of sucrose and detergents

Purified plasma membrane vesicles were isolated in the presence of 250 mM sucrose from 7-day-old roots of Triticum aestivum L. cv. Drabant by aqueous polymer two-phase partitioning. When added to a low-salt medium containing 9-aminoacridine (9-AA), the vesicles caused a much larger total decrease in 9-AA fluorescence when sucrose was absent than when sucrose was present. A slow component of the decrease was also larger in the absence of sucrose. Triton X-100 reduced the decrease in 9-AA fluorescence upon vesicle addition and abolished completely the slow component of the decrease. There was no correlation between the time-dependence of 9-AA fluorescence and that of the Mg²⁺-ATPase described below. The time course of Mg²⁺-ATPase activity was followed by sampling at short intervals (down to 10 s) and analyzing for P, released. In the absence of detergent, the rates of P, release were linear from zero minutes, whether 250 mM sucrose was present or not, but the rate was 10^?50% higher in the absence of sucrose than in its presence. Sucrose (250 mM) added during a minus-sucrose assay lowered Mg²⁺-ATPase activity within 2 min to the level observed with 250 mM sucrose present from the start. The effect of 25-1 100 mM sucrose was tested and there was little or no effect below KM) mM. Above 100 mM sucrose the rate of P, release decreased drastically; at 1 100 mM sucrose the rate was ca 20% the rate at 25 mM sucrose. The inhibitory effect of sucrose was not alleviated by increased concentrations of Mg²⁺ and/or ATP. nor was it affected by the presence or absence of Triton X-100. We conclude that sucrose somehow inhibits the Mg²⁺-ATPase directly or affects the conformation of the plasma membrane in such a way as to inhibit the enzyme. The presence of detergents increased Mg²⁺-ATPase activity in the order Triton X-100 (4–5-fold) > Zwittergent 3–14 = Na-cholate = octylglucoside > digitonin (2-fold). In all cases optimal activity was observed at detergent concentrations at or below the critical micellar concentration. The detergent concentration curves could be simulated by the sum of a stimulatory and an inhibitory reaction. At the optimal concentration, digitonin gave a linear time-course of P, release, whereas all the other detergents showed a distinct lag of 1–3 min before maximal rates were attained. The problems of using detergents in polarity assays are discussed.     

Prediction of heavy-metal concentrations in mature plants by chemical analysis of seedlings

Five plant species were cultivated on a soil from the Neckar alluvial fan near Heidelberg (FRG) polluted by the emissions of a cement plant. Thallium, cadmium and lead concentrations in seedlings and mature plants were determined by atomic absorption analysis. AdditionallyBrassica napus L.napus was grown on soils containing 5 different concentrations of heavy metals, achieved by mixing two similar soils, from the same area but with different metal concentrations. Thallium and cadmium were shown to be taken up by roots whilst lead which was also absorbed, was deposited mainly on the plant surface. However during cultivation in the winter months, a remarkable deposit of lead via the roots was found. Thallium in the soil from a anthrorogen source was more available to plants than thallium of geological origin. During the lifetime of a plant concentrations of thallium and cadmium were always highest in the seedling. The decrease in metal concentration with maturity depended on the plant species and the element, but was not a function of the metal concentration in the soil.     

C-banding polymorphism and the analysis of nucleolar activity in Dasypyrum villosum (L.) Candargy,its added chromosomes to hexaploid wheat and the amphiploid Triticum dicoccum — D. villosum

Summary C-banding patterns and nucleolar activity were analyzed in Dasypyrum villosum, its added chromosomes to hexaploid wheat and the hexaploid amphiploid Triticum dicoccum-D. villosum. Two different populations of the allogamous species D. villosum (2n= 14, VV) from Greece and Italy were analyzed showing a similar polymorphism for C-banding pattern. Six of the seven addition lines were identified by their characteristic C-banding pattern. No polymorphism between both members of each added alien chromosome was found. Furthermore, nucleolar activity and competition were studied by using silver staining procedure. In D. villosum only one chromosome pair, A, was found to be responsible for organizing nucleoli. The results obtained in the amphiploid and in the addition lines demonstrate that nucleolar activity is restricted to SAT-chromosomes 1B and 6B of wheat, while those of D. villosum remain inactive.