Free sterols and glycolipids in the aleurone tissue of germinating wheat

The action of gibberellic acid on wheat aleurone tissue led to reduced levels of free sterols and glycolipids compared with control tissue. Radio-labelled precursors were not incorporated into sterols or glycolipids, although mevalonate and glycerol were incorporated into polyisoprenoid hydrocarbon and phospholipid respectively. It is concluded that sterols and glycolipids are not synthesized in the tissue during germination; this is in contrast to earlier reports of the active, gibberellin-regulated metabolism of phospholipids.     

Characterisation of germinating and non-germinating wheat seeds by nuclear magnetic resonance (NMR) spectroscopy

Experiments were conducted to characterise the changes, especially of water status in germinating and non-germinating wheat seeds by nuclear magnetic resonance (NMR) spectroscopy. NMR relaxation time (T₂) measurements showed tri-phasic or bi-phasic characteristics during different stages of hydration, depending on the seed's ability to germinate. Component analysis of T₂ data revealed the existence of only two components, bound and bulk water, in dry seeds. In contrast, both the germinating and non-germinating wheat seeds had a three-component water proton system (bound, bulk and free water) in phase I of hydration. During the lag phase (phase II) of hydration, bulk water component of non-germinating seeds disappeared completely, resulting in a two component water proton system. Nevertheless, the three component water proton system was observed in the germinating seeds in phase II. Following phase II, rapid hydration (phase III) was observed in germinating seeds only. Water protons were re-organised and there were increases in bulk and free water but decreases in bound water concomitantly. Comparison of the physical state of water in these seeds by NMR spectroscopy with that of tissue leachate conductivity measurement suggests that the seed membrane system was affected more evidently in non-germinating seeds, leading to the disorganised cell structure. The present study provides evidence that the reorganisation of physical state of water in germinating wheat seeds during hydration is essential for its subsequent event of germination.     

Resistance of wheat aleurone cell walls to acid and xylanase action

Isolated wheat (Triticum aestivum var. Potam) aleurone layers have a high capacity to acidify their environment, and secrete hydrolytic enzymes (endoxylanase, glucanase, α-amylase, proteases, etc.) under the control of GA₃. Acidic pH and xylanases are found to be essential for cell wall relaxation in growing tissues, but aleurone is a non-growing, non-dividing tissue. In this tissue, we studied the effect of these loosening factors on aleurone cell walls.Exposure to pH 3.0 caused the release of carbohydrates and calcium ions from the pericarp, and a small amount of carbohydrates, mainly polysaccharides, from aleurone layers from which pericarp tissue had been removed. 50 percnt; of the total sugars released into the incubation medium by these isolated aleurone tissue was arabinose, but no xylose, calcium ions, or phenolic compounds were found. Acid preincubation decreased by 30 percnt; the susceptibility of aleurone cell walls to degradation by exogenously-applied endoxylanase, and also modified the architecture of cell wall as observed by autofluorescence of phenolic groups. These findings suggest that acid treatment and endoxylanase action, rather than having a loosening effect on aleurone cell wall, can have an opposite effect, increasing the resistance of aleurone cell walls to loosening.     

Hormones-induced modifications in the response of wheat flag leaf to NaCl

A field experiment was carried out to investigate the effects of presoaking the wheat grains (Triticum aestivum L.) in 33 or 66 mM NaCl and indolyl-3-acetic acid (IAA at 50 g m⁻³), gibberellic acid (GA₃ at 100 g m⁻³) or kinetin (100 g m⁻³) on some tolerance criteria in wheat flag leaf at different stages of development. At various stages of flag leaf development pretreatment with 33 or 66 mM NaCl decreased degree of succulence (particularly 66 mM), relative growth rate, net assimilation rate, relative water content, K⁺ content and K⁺/Na⁺ ratio and at the same time induced accumulation of abscisic acid and Na⁺. In the majority of cases grain pretreatment with GA₃ or kinetin and to a lesser extent with IAA alleviated either partially or completely the deleterious effect of salinity on the above mentioned parameters.     

Changes in phytohormone contents in chickpea seeds germinating under lead or zinc stress

The present work describes the changes that take place in phytohormone contents in germinating chickpea (Cicer arietinum cv. Aziziye-94) seeds in response to heavy metal stress. For this aim, endogenous abscisic acid (ABA), gibberellic acid (GA₃), zeatin (Z) and zeatin riboside (ZR) contents were followed for 24, 48 and 72 h in chickpea seeds germinating at the concentrations of 0.1, 1.0 and 5.0 mM Pb or 0.1, 1.0 and 10 mM Zn. The results showed that Pb and Zn significantly delayed and impeded the germination of chickpea seeds. The negative effect of Pb on germination was higher than that of Zn. Further, Pb increased ABA and Z contents while decreased GA₃ content in the germinating seeds. The high concentrations of Zn (1.0 and 10 mM) decreased contents of Z, ZR and GA₃ while 0.1 mM Zn increased the content of the same hormones. The ABA content was enhanced by Zn in all concentrations used.     

A comparison of methods for delivering DNA to wheat: the application of wheat dwarf virus DNA to seeds with exposed apical meristems

The apical meristems in dry wheat seeds were exposed (dissected seeds) to provide a target for DNA uptake. Using wheat, dwarf virus as the marker DNA, various methods of delivery were compared. Dry dissected seeds imbided in wheat dwarf virus DNA solution gave infection in 16% of the seedlings growing from them. A wheat dwarf virus dimer placed between the T-DNA borders of a vector plasmid inAgrobacterium tumefaciens (disarmed C58) andA. rhizogenes (LBA 9402) gave high levels of infection (79%) when dissected seeds were soaked in theAgrobacterium inoculum (agroinfection). Bombardment of dry dissected seeds with tungsten particles coated in wheat dwarf virus DNA did not give infection, but when softened by presoaking in water for 14 h, infection was observed at a low level (3%). The exposure of the apical meristem in all three methods gave a higher frequency of infection compared with treating intact seeds and in some cases the difference was substantial. The significance of the approach for DNA uptake studies is discussed along with its relevance to achieving stable transformation with non-viral constructs.     

Production of polyclonal antibodies against a wheat thioredoxinh by genetic immunization

The recent techniques of genetic immunization, in which DNA constructs are introduced directly into mammalian tissuesin vivo, were used to produce antisera against thioredoxinh, a protein of wheat. Two rabbits and two mice were inoculated intramuscularly with a vector containing the cDNA encoding the protein of interest under control of a cytomegalovirus promoter. No immune response was observed in rabbits, even when a fourfold quantity of DNA and a different inoculation site were used. By contrast, an inoculated mouse was found to produce antisera against the wheat thioredoxinh as analyzed by western blotting. This technique appears useful, therefore, to obtain polyclonal antibodies against plant proteins that are difficult to purify, if the corresponding cDNAs are available.     

Chromosomal localization of structural genes and regulators in wheat by 2D electrophoresis of ditelosomic lines

Summary Among the 782 spots observed in two-dimensional gel electrophoresis of denatured proteins from etiolated wheat shoots, 185 were found to be variable between the euploid and 26 ditelosomic lines of Chinese Spring. Thirty-five structural genes were located on 17 chromosome arms. Numerous intensity changes showing alterations in protein levels were observed and led to the following statements: 1) regulators are frequently found and can be assigned for a same polypeptide to various chromosome arms; 2) for most polypeptides homoeologous arms do not manifest similar effects; 3) nevertheless, when affecting the same polypeptide, homoeologous arms display in most cases identical regulatory effects; 4) gene dosage compensation is observed in only one out of four homoeoallelic situations.     

Cytological analysis on the distribution and origin of the alien chromosome pair conferring blue aleurone color in several European common wheat (Triticum aestivum L.) strains

Summary Meiotic chromosome pairing and Giemsa C-banding analyses in crosses of several European blue-grained wheat strains with Chinese Spring double ditelosomic and other aneuploid lines showed that Triticum aestivum Blaukorn strains Berlin, Probstdorf, Tschermak, and Weihenstephan are chromosome substitutions, in which the complete wheat chromosome 4A pair is replaced, whereas the strains Brünn and Moskau are 4B substitutions. The alien chromosome pair in all of these strains is an A genome chromosome (4A) from diploid Triticum monococcum or T. boeoticum not present in common tetraploid and hexaploid cultivated wheats. The Blaukorn strain Weihenstephan W 70a86 possesses, in addition to a rye chromosome pair 5R compensating for the loss of part of chromosome 5D, a 4A/5DL translocation replacing chromosome pair 4B of wheat.     

Contribution of aleurone layer and scutellum to α-amylase synthesis and secretion in wheat and rice grains

Intact aleurone layers from wheat ( Triticum aestivum L. cv Camp Rémy) and rice ( Oryza sativa L. cv Cigalon) grains both contained and secreted more α-amylase (EC 3.2.1.1.) than did the corresponding scutellar tissues. This discrimination was already evident at the earliest stages of germination at which the tissues could be isolated, and became more pronounced upon subsequent germination and growth. Isoenzyme patterns obtained upon isoelectric focusing showed a considerable polymorphism of the α-amylases of each cereal. The enzyme polymorphism pattern was the same in the aleurone layer and in the scutellum, but some secondary constituents appeared to be more specific for the one or the other of the tissues. Moreover, the isozymes found in the tissues were the same as those found to be secreted. A third α-amylase antigen which differs from the well established α₁ and α₁₁ forms was identified in the germinating wheat grains. The presence of Ca²⁺ in the secretion medium favoured maximum secretion of α-amylases from the wheat scutellum and aleurone layers, whereas it inhibited the secretion of the enzymes from the rice aleurone layer.     

Identification and localization of molecular markers linked to the Lr9 leaf rust resistance gene of wheat

Near-isogenic lines (NILs) for the leaf rust resistance gene Lr9 were screened for polymorphisms at the molecular level. RAPD (random amplified polymorphic DNA) primers as well as RFLP (restriction fragment length polymorphism) markers were used. Out of 395 RAPD primers tested, three showed polymorphisms between NILs, i.e., an additional band was found in resistant lines. One of these polymorphic bands was cloned and sequenced. Specific primers were synthesized, and after amplification only resistant lines showed an amplified product. Thus, these primers define a sequence-tagged site that is specific for the translocated fragment carrying the Lr9 gene. A cross between a resistant NIL and the spelt (Triticum spelta) variety Oberkulmer was made, and F₂ plants were analyzed for genetic linkage. All three polymorphisms detected by the PCR (polymerase chain reaction) and one RFLP marker (cMWG684) showed complete linkage to the Lr9 gene in 156 and 133 plants analyzed, respectively. A second RFLP marker (PSR546) was closely linked (8±2.4 cM) to the Lr9 gene and the other four DNA markers. As this marker maps to the distal part of the long arm of chromosome 6B of wheat, Lr9 and the other DNA markers also map to the distal region of 6BL. All three PCR markers detected the Lr9 gene in independently derived breeding lines and varieties, thus proving their general applicability in wheat breeding programs.     

Isolation and analysis of endophytic microorganisms in wheat (Triticum aestivum L.) leaves

The present investigation was undertaken in order to select the surface-sterilization technique most efficient for eliminating epiphytes, to document the spectrum of endophytes of healthy leaves from three wheat cultivars in Buenos Aires Province (Argentina) and to determine their infection frequencies at three growth stages. Surface-sterilization with undiluted commercial solution of sodium hypochlorite was reaffirmed as adequate for removing epiphytes on wheat leaves. From the 450 wheat leaf segments incubated, three bacterial isolates and 130 fungal isolates were obtained. From all the isolates, 19 fungal species were identified. Bacterial isolates were characterized as Bacillus sp. There were significant differences between microorganisms, stages of growth, and stages × microorganisms interaction. Differences between cultivars, stages × cultivars, microorganisms × cultivars and for the triple interaction were not significant. Frequency of microorganisms isolated increased with crop age, but it was statistically similar for the three wheat cultivars tested (Klein Centauro, Klein Dragón and Buck Ombú). Rhodotorula rubra, Alternaria alternata, Cladosporium herbarum and Epicoccum nigrum were isolated in the highest frequency. The other microorganisms were present at intermediate or low values. The species isolated may be assigned to three groups: (a) well-known and economically important pathogens of wheat, (b) commonly abundant phylloplane fungi considered to be primary saprobic and minor pathogens and (c) species occasionally present in wheat.     

The control of boron accumulation by two genotypes of wheat

The mechanism of boron (B) uptake in wheat was studied using two genotypes with known differences in their ability to accumulate B. Influx and efflux of B was measured in the roots of intact 21 d old plants.Roots grown in 15 M B, when transferred to solutions containing 1mM B showed a rapid increase in B content for up to 60 min, after which no further increase was evident up to 4 h. No genotypic difference in B influx was apparent over these time periods. Roots grown in 1mM B for 7 d and then rinsed in B-free solutions quickly lost most of B that they contained within 1 hour; little further efflux was observed over the following three hours. As with the influx, no genotypic difference in B flux was evident.It is suggested that the lack of genotypic difference in the short-term B fluxes could be due to a masking effect of extracellular B bound in the cell walls of the roots.Department of Botany, University of Adelaide     

Wheat germ agglutinin (WGA) gene expression and ABA accumulation in the developing embryos of wheat (Triticum aestivum) in response to drought

Expression of wheat germ agglutinin (WGA) gene inthe developing embryos of wheat (Triticumaestivum L. cv. C-306) was studied in relation toabscisic acid (ABA) accumulation under water stressconditions. Imposition of water stress resulted inelevated ABA levels in the embryos at threedevelopmental stages (18, 24 and 30 DPA). On thecontrary, the effect of drought stress on WGAaccumulation was stage dependent with significantincrease in WGA content being observed at only 24 DPA. Our results suggest that apart from ABA, otherfactors which are temporally expressed, are alsoinvolved in regulation of WGA gene expression.     

Regulatory effects of homoeologous chromosome arms on wheat proteins at two developmental stages

Summary Two-dimensional gel electrophoresis was conducted on denatured proteins of the 10-day-old first leaf (1F stage) of 18 homoeologous ditelosomic (DT) lines of wheat cultivar Chinese Spring. The observations, compared to the euploid control and relative to previous data found on 7-day-old etiolated seedlings (G7 stage) of the same lines lead to the following statements: 1) the structural genes of 24 spots can be assigned to 12 chromosome arms; 2) regulatory effects are completely different between the 1F and the G7 stages which may indicate that the regulation of protein amounts is often stage-specific; 3) no case of complete gene dosage compensation is observed among 4 groups of hypothesized homoeoallelic products; 4) homoeologous DT lines do not manifest similar effects which suggest the absence of homoeology for the detected regulatory effects.     

Modulation of somatic embryogenesis in early and late-stage embryos of wheat (Triticum aestivum L.) under the influence of (±)-abscisic acid and its analogs

Zygotic embryos from ten spring wheat (Triticum aestivum L.) genotypes were tested for embryogenic callus induction in the presence or absence of externally supplied (±)-abscisic acid (ABA) and two of its analogs, methyl abscisate and methyl epoxy-beta-ionylideneacetate. (±)-ABA and its analogs suppressed precocious germination of cultured late-stage embryos and promoted embryogenic callus induction. A significantly greater number of plants was regenerated from calli induced in the presence of ABA and ABA analogs. Early-stage embryos when cultured in the presence of (±)-ABA showed a negative response. Possible roles of ABA with respect to the expression of somatic embryogenesis are discussed.Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Growth and cell wall properties of two wheat cultivars differing in their sensitivity to aluminum stress

The present study was conducted to investigate the cell wall properties in two wheat (Triticum aestivum L.) cultivars differing in their sensitivity to Al stress. Seedlings of Al-resistant, Inia66 and Al-sensitive, Kalyansona cultivars were grown in complete nutrient solutions for 4 days and then subjected to treatment solutions containing Al (0, 50 microM) in a 0.5 mM CaCl(2) solution at pH 4.5 for 24 h. Root elongation was inhibited greatly by the Al treatment in the Al-sensitive cultivar compared to the Al-resistant cultivar. The Al-resistant cultivar accumulated less amount of Al in the root apex than in the Al-sensitive cultivar. The contents of pectin and hemicellulose in roots were increased with Al stress, and this increase was more conspicuous in the Al-sensitive cultivar. The molecular mass of hemicellulosic polysaccharides was increased by the Al treatment in the Al-sensitive cultivar. The increase in the content of hemicellulose was attributed to increase in the contents of glucose, arabinose and xylose in neutral sugars. Aluminum treatment increased the contents of ferulic acid and p-coumaric acid especially in the Al-sensitive cultivar by increasing the activity of phenylalanine ammonia lyase (PAL, EC 4.3.1.5). Aluminum treatment markedly decreased the beta-glucanase activity in the Al-sensitive cultivar, but did not exert any effect in the Al-resistant cultivar. These results suggest that the modulation of the activity of beta-glucanase with Al stress may be involved in part in the alteration of the molecular mass of hemicellulosic polysaccharides in the Al-sensitive cultivar. The increase in the molecular mass of hemicellulosic polysaccharides and ferulic acid synthesis in the Al-sensitive cultivar with Al stress may induce the mechanical rigidity of the cell wall and inhibit the elongation of wheat roots.     

Chromosome substitutions of Triticum timopheevii in common wheat and some observations on the evolution of polyploid wheat species

Whether the two tetraploid wheat species, the well known Triticum turgidum L. (macaroni wheat, AABB genomes) and the obscure T. timopheevii Zhuk. (A^tA^tGG), have monophyletic or diphyletic origin from the same or different diploid species presents an interesting evolutionary problem. Moreover, T. timopheevii and its wild form T. araraticum are an important genetic resource for macaroni and bread-wheat improvement. To study these objectives, the substitution and genetic compensation abilities of individual T. timopheevii chromosomes for missing chromosomes of T. aestivum Chinese Spring (AABBDD) were analyzed. Chinese Spring aneuploids (nullisomic-tetrasomics) were crossed with a T. timopheevii x Aegilops tauschii amphiploid to isolate T. timopheevii chromosomes in a monosomic condition. The F₁ hybrids were backcrossed one to four times to Chinese Spring aneuploids without selection for the T. timopheevii chromosome of interest. While spontaneous substitutions involving all A^t- and G-genome chromosomes were identified, the targeted T. timopheevii chromosome was not always recovered. Lines with spontaneous substitutions from T. timopheevii were chosen for further backcrossing. Six T. timopheevii chromosome substitutions were isolated: 6A^t (6A), 2G (2B), 3G (3B), 4G (4B), 5G (5B) and 6G (6B). The substitution lines had normal morphology and fertility. The 6A^t of T. timopheevii was involved in a translocation with chromosome 1G, resulting in the transfer of the group-1 gliadin locus to 6A^t. Chromosome 2G substituted for 2B at a frequency higher than expected and may carry putative homoeoalleles of gametocidal genes present on group-2 chromosomes of several alien species. Our data indicate a common origin for tetraploid wheat species, but from separate hybridization events because of the presence of a different spectrum of intergenomic translocations.     

Genotypic differences in phosphorus efficiency of wheat

In an attempt to evaluate whether breeding and selection for high yielding capacity did change the P requirements of modern wheat cultivars, the response of two wheat cultivars to different levels of P supply was investigated. A traditional cultivar ("Peragis") and a modern cultivar ("Cosir") were cultivated in a C-loess low in available P and high in CaCO₃ in 120 cm high PVC pots. Shoot and root growth at different developmental stages was compared. The grain yield of the modern cultivar Cosir was higher at limiting and non-limiting P supply and, therefore, this cultivar can be considered as more P-efficient than the traditional cultivar. From the results it can be concluded that the main factors contributing to the higher P efficiency of the modern cultivar are (i) efficient use of assimilates for root growth characteristics which enhance P acquisition: smaller root diameter, and longer root hairs, (ii) efficient remobilization of P from vegetative plant organs to the grains, and (iii) lower P requirement for grain yield formation because of lower ear number per plant but higher grain number per ear.     

Amino acids and quaternary nitrogen compounds in the germinating wheat grain

The tissues of the quiescent wheat grain contained free amino acids and quaternary nitrogen compounds. During germination the amino acid levels increased several fold. In the aleurone tissue and starchy endosperm glutamine was the predominant amino acid. Asparagine was predominant in the seedling tissues. Choline and glycine betaine were the principal quaternary nitrogen compounds present. The aleurone tissue and the embryo/seedling contained large quantities of glycine betaine. The increase in free amino acid levels in the aleurone tissue during the first 2 days of germination occurred independently of the embryo. After the second day, the further increase in levels was dependent upon the presence of the embryo and of gibberellic acid (GA). Estimation of the individual amino acids and quaternary nitrogen compounds released from incubating aleurone layers into aqueous media revealed a selective release of some compounds and retention of others. The process was regulated by GA. Possible mechanisms for the release of amino acid and its control by GA are discussed.