Characterization of cytosolic phosphoglucoisomerase from immature wheat (Triticum aestivum L.) endosperm

Phosphoglucoisomerase from cytosol of immature wheat endosperm was purified 650-fold by ammonium sulphate fractionation, isopropyl alcohol precipitation, DEAE-cellulose chromatography and gel filtration through Sepharose CL-6B. The enzyme, with a molecular weight of about 130,000, exhibited maximum activity at pH 8.1. It showed typical hyperbolic kinetics with both fructose 6-P and glucose 6-P withK _m of 0.18 mM and 0.44mM respectively. On either side of the optimum pH, the enzyme had lower affinity for the substrates. Using glucose 6-P as the substrate, the equilibrium was reached at 27% fructose 6-P and 73% glucose 6-P with an equilibrium constant of 2.7. The ΔF calculated from the apparent equilibrium constant was +597 cal mol^-1. The activation energy calculated from the Arrhenius plot was 5500 cal mol^-1. The enzyme was completely inhibited by ribose 5-P, ribulose 5-P and 6-phosphogluconate, withK _i values of 0.17, 0.25 and 0.14 mM respectively. The probable role of the enzyme in starch biosynthesis is discussed.     

The chromosomal location of peroxidase isozymes of the wheat kernel

Summary The analysis of the individual parts of the Triticum aestivum L. kernel yields a total of 11 peroxidase isozymes: m, n, a, c, d₁, d, d₂, e, f, g and h (in order from faster to slower migration). Isozymes a, c and d are found in the endosperm (Ed) and seed coats (C), while m, n, d₁, d₂, e, f, g and h are peculiar to the embryo and scutellum (E + S). The use of the nullitetrasomic and ditellosomic series of Chinese Spring wheat allows peroxidase isozymes to be associated with specific chromosome arms. Isozymes a, c and d (Ed) are associated with chromosome arms 7D^S, 4B^L and 7A^S; whereas isozymes m, d₂, e and f are associated with chromosome arms 3D^S, 3B^L, 3D^L and 3D^L, respecitvely. Thus, the E + S isozymes are associated with homoeology group 3 and the Ed isozymes with homoeology groups 7 (a and d isozymes) or 4 (c isozymes).     

Expression of stress proteins (HSP-70 and HSP-90) in the rabbit urinary bladder subjected to partial outlet obstruction

Partial obstruction of the rabbit bladder outlet induces a rapid hypertrophy characterized by increased bladder mass, increased smooth muscle content, and increased collagen deposition. In addition, partial outlet obstruction induces decreased contractile responses to both field stimulation and postsynaptic receptor stimulation. Although the morphological and contractile responses to partial outlet obstruction have been well characterized, there is little information on the cellular and molecular mechanisms of these changes. In a previous study, we demonstrated that one of the earliest genes to be expressed following partial outlet obstruction in rabbits was the gene expressing stress protein-70 (HSP-70). In order to further define the genetic and molecular basis of these responses, the expression of stress gene products HSP-70 and HSP-90 in rabbit urinary bladder subjected to partial outlet obstruction has been quantitatively evaluated by Western blot coupled with laser densitometry using anti-HSP-70 and-90 monoclonal antibodies. The data show that stress gene products HSP-70 and HSP-90 are constitutively expressed in control rabbit bladder tissue and transiently increased following partial outlet obstruction. Increased content of HSP-70 was detected at 6 hr after obstruction and reached a maximum (2.7-fold over the control level) at 24 hr. Increased HSP-90 was also detected at 6 hr but reached a maximum (4.5-fold over the control level) at 12 hr. By 7 day post-obstruction, the content of these two proteins returned to the control levels. This study suggests that alterations of stress gene expression resulting in increased HSP-70 and 90 may play an important role in the response of the bladder to partial outlet obstruction.     

Molecular analysis of plants regenerated from embryogenic cultures of wheat (Triticum aestivum L.)

Total DNAs of plants regenerated from immature embryo-derived 2-month-old embryogenic calli of wheat (cultivars Florida 302, Chris, Pavon, RH770019) were probed with six maize mitochondrial genes (atpA, atp6, apt9, coxI, coxII, rrn18-rrn5), three hypervariable wheat mitochondrial clones (K, K3, X2), five random pearl millet mitochondrial clones (4A9, 4D1, 4D12, 4E1, 4E11) and the often-used wheat Nor locus probe (pTA71), in order to assess the molecular changes induced in vitro. In addition, protoplast-derived plants, and 24-month-old embryogenic and non-embryogenic calli and cell suspension cultures of Florida 302 were also analyzed. No variation was revealed by the wheat or millet mitochondrial clones. Qualitative variation was detected in the nonembryogenic suspension culture by three maize mitochondrial genes (coxI, rrn18-rrn5, atp6). A callus-specific 3.8-kb Hind III fragment was detected in all four cultivars after hybridization with the coxI gene. The organization of the Nor locus of the plants regenerated from Florida 302 and Chris was stable when compared to their respective control plants and calli. The Nor locus in regenerants of Pavon and RH, on the other hand, was found to be variable. However, Nor locus variability was not observed in 14 individual seed-derived control plants from either Pavon or RH sources. In Pavon, a 3.6-kb Taq I or a 5.6-kb Bam HI+ Eco RI fragment was lost after regeneration. In one of the RH regenerants, which lost a fragment, an additional fragment was observed.     

Inheritance of grain proteins in wheat

Summary Diallel crosses between five divergent vulgare wheat cultivars were made in order to evaluate the mode of inheritance and combining ability of grain proteins. Significant differences in grain protein content were found between cultivars and their hybrids. It was established that the inheritance of seed protein in the F₁ generation included both additive and non-additive gene action.     

The endophytic fungi from wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

In order to study the species composition of endophytes from wheat healthy plants in Buenos Aires Province (Argentina) and to determine their infection frequencies from leaves, stems, glumes and grains, wheat plants were collected from five cultivars at five growth stages from crop emergence to harvest. A total of 1,750 plant segments (leaves, stems, glumes and grains) were processed from the five wheat cultivars at five growth stages, and 722 isolates of endophytic fungi recovered were identified as 30 fungal genera. Alternaria alternata, Cladosporium herbarum, Epicoccum nigrum, Cryptococcus sp., Rhodotorula rubra, Penicillium sp. and Fusarium graminearum were the fungi that showed the highest colonization frequency (CF%) in all the tissues and organs analysed. The number of taxa isolated was greater in the leaves than those in the other organs analysed.     

Identification of callus types for long-term maintenance and regeneration from commercial cultivars of wheat (Triticum aestivum L.)

Summary Immature embryos, inflorescences, and anthers of eight commercial cultivars of Triticum aestivum (wheat) formed embryogenic callus on a variety of media. Immature embryos (1.0–1.5 mm long) were found to be most suitable for embryogenic callus formation while anthers responded poorly; inflorescences gave intermediate values. Immature embryos of various cultivars showed significant differences in callus formation in response to 11 of the 12 media tested. No significant differences were observed when the embryos were cultred under similar conditions on MS medium with twice the concentration of inorganic salts, supplemented with 2,4-D, casein hydrolysate and glutamine. Furthermore, with inflorescences also no significant differences were observed. Explants on callus formation media formed two types of embryogenic calli: an off-white, compact, and nodular callus and a white compact callus. Upon successive subcultures (approximately 5 months), the nodular embryogenic callus became more prominent and was identified as aged callus. The aged callus upon further subculture, formed an off-white, soft, and friable embryogenic callus. Both the aged and friable calli maintained their embryogenic capacity over many subculture passages (to date up to 19 months). All embryogenic calli (1 month old) from the different callus-forming media, irrespective of expiant source, formed only green shoots on regeneration media that developed to maturity in the greenhouse. There were no significant differences in the response of calli derived from embryos and inflorescences cultured on the different initiation media. Also, the shoot-forming capacity of the cultivars was not significantly different. Anther-derived calli formed the least shoots. Aged and friable calli on regeneration media also formed green shoots but at lower frequencies. Plants from long-term culture have also been grown to maturity in soil.Florida Agricultural Experiment Station Journal Series No. R-00494     

An improved medium for in vitro grain development from detached wheat spikelets

Wheat spikelets detached from the spike at anthesis were cultured on solidified media and successfully produced mature grains. These grains resembled normal grains and contained well-developed, embryos. Lower concentrations of glutamine favored dry weight increase in developing grains. Such grains were indistinguishable from grains from greenhouse-grown plants in germination on moist blotting sheets. The technique of individual spikelet culture can be used to study physiology and development of wheat grains and kernels and to study host-pathogen interactions in wheat floret diseases such as Karnal bunt.     

Genetic and environmental considerations for evaluating crown position of wheat

Summary Crown position affects winter survival of fallsown wheat (Triticum aestivum L.) Direct or indirect selection for crown depth has been little practiced. Reports have suggested that short subcrown internode length was closely related to semidwarf plant height and that semidwarfism was related to poor emergence. This study determined the relationships among crown depth, plant height, and emergence rate index in three wheat populations. The efficiency of evaluating crown placement in the field was examined and additional information was obtained on its genetic control. The F₂-derived F₄ and F₅ lines from the crosses of female parents Daws, Nugaines, and Stephens with male parent Selection 7952 were planted at Central Ferry and Pullman, Washington, respectively. Correlations from each population indicated that crown depth and subcrown internode length were not closely associated with plant height and emergence rate index. Crown depth was a more reliable indicator of crown placement than subcrown internode length. Adjustment of the data for seed depth differences was essential for evaluating subcrown internode length but less important for evaluating crown depth. After adjustment for seed depth, narrow-sense h ² values for subcrown internode length and crown depth were 0.25–0.41. Crown depth and subcrown internode length were inherited as quantitative traits in phenotypes that expressed variable dominance. Modest gains due to selection for crown depth were achieved.Contribution from USDA-ARS and College of Agriculture and Home Economics Research Center, Washington State University, Scientific Paper No. 7795     

Molecular characterization of the fate of transgenes in transformed wheat (Triticum aestivum L.)

Molecular analysis of the transgenes bar and gus was carried out over successive generations in six independent transgenic lines of wheat, until the plants attained homozygosity. Data on expression and integration of the transgenes is presented. Five of the lines were found to be stably transformed, duly transferring the transgenes to the next generation. The copy number of the transgenes varied from one to five in the different lines. One line was unstable, first losing expression of and then eliminating both the transgenes in R3 plants. Although the gus gene was detected in all the lines, GUS expression had been lost in R2 plants of all but one line. Rearrangement of transgene sequences was observed, but it had no effect on gene expression. All the stable lines were found to segregate for transgene activity in a Mendelian fashion.     

Yield response of various wheat genotypes to inoculation withAzospirillum brasilense

Summary Twenty different wheat genotypes representing a wide genetic variability, were tested for their yield and yield components response to inoculation withAzospirillum brasilense, at two levels of N fertilization. Only two cultivars responded by a significant yield increase of 7.4 and 8.0 per cent — both at the higher N level. The response reflected an increase in the number of grains per plant added as additional spikes. The importance of the host plant genotype for a successful wheat-bacteria association is discussed.     

An efficient method for in vitro regeneration from immature inflorescence explants of Canadian wheat cultivars

Fertile, green plants were regenerated from immature inflorescence explants from each of four Canadian wheat cultivars. The cultivars were representative of four classes of Canadian wheat. Explants from immature inflorescences of three size ranges were cultured on two types of media: MSI/MSR, which contains 1650 mg l^-1 NH₄NO₃and sucrose as a carbon source, and BII/BIR, which contains 250 mg l^-1 NH₄NO₃and maltose as a carbon source. Regeneration from all cultivars was significantly better on BII/BIR media than on MSI/MSR media. On BII/BIR media, `AC Karma', `Plenty', and `Fielder' gave the highest number of shoots per 10 explants, where the explants were derived from immature inflorescences 5.1 to 10.0 mm in length. 'Columbus' did not regenerate on MSI/MSR medium, and regenerated poorly on BII/BIR medium. Differences were found between cultivars with regard to the number of regenerant plants produced with the best treatments: `Plenty' produced 16.1 shoots per 10 explants, `AC Karma' 12.4, `Fielder' 6.4, and `Columbus' 2.2.     

Characterization of the expression of a wheat cystatin gene during caryopsis development

A cDNA coding for phytocystatin, a protease inhibitor, was isolated from wheat embryos by differential display RT-PCR and the corresponding full-length cDNA (named WC5 for wheat cystatin gene 5) subsequently obtained by RACE. The deduced primary sequence of the protein suggests the presence of a 28 amino acid N-terminal signal sequence and a 100 amino acid mature protein containing the three consensus motifs known to interact with the active site of cysteine peptidases. Northern and western analysis revealed a spatio-temporal pattern of the cystatin gene expression during caryopse development. In the embryo, WC5 was only expressed during early embryogenesis whereas, in seed covering layers, WC5 expression was restricted to the maturation stage of grain development. In addition, immunolocalization experiments showed that cystatin accumulated in the aleurone layer of the maturating seed and in the parenchymal tissues of the embryo scutellum. A recombinant form of the wheat cystatin was shown to be able to inhibit peptidase activities present in whole seed protein extracts. In addition, immunological techniques allowed us to identify two putative target peptidases. The possible roles of the cystatin protein are discussed in relation with tissular localization and putative peptidase targets during seed maturation.     

RELP markers linked to two Hessian fly-resistance genes in wheat (Triticum aestivum L.) from Triticum tauschii (coss.) Schmal

Summary Restriction fragment length polymorphism (RFLP) markers linked to genes controlling Hessian fly resistance from Triticum tauschii (Coss.) Schmal. were identified for two wheat (Triticum aestivum L.) germ plasm lines KS89WGRC3 (C3) and KS89WGRC6 (C6). Forty-six clones with loci on chromosomes of homoeologous group 3 and 28 clones on those of group 6 were surveyed for polymorphisms. Eleven and 12 clones detected T. tauschii loci in the two lines, respectively. Analysis of F₂ progenies indicated that the Hessian fly resistance gene H23 identified in C3 is linked to XksuH4 (6.9 cM) and XksuG48 (A) (15.6 cM), located on 6D. The resistance gene H24 in C6 is linked to XcnlBCD451 (5.9 cM), XcnlCD0482 (5.9 cM) and XksuG48 (B) (12.9 cM), located on 3DL.Paper No. 810 of the Cornell Plant Breeding Series     

Enzyme activities associated with developing wheat(Triticum aestivum L.) grain amyloplasts

Intact amyloplasts from endosperm of developing wheat grains have been isolated by first preparing the protoplasts and then fractionating the lysate of the protoplasts on percoll and ficoll gradients, respectively. Amyloplasts isolated as above were functional and not contaminated by cytosol or by organelles likely to be involved in carbohydrate metabolism. The enzyme distribution studies indicated that ADP-glucose pyrophosphorylase and starch synthase were confined to amyloplasts, whereas invertase, sucrose synthase, UDP-glucose pyrophosphorylase, hexokinase, phosphofructokinase-2 and fructose-2,6-P₂ase were absent fro the amyloplast and mainly confined to the cytosol. Triose-P isomerase, glyceraldehyde-3-P dehydrogenase, phosphohexose isomerase, phosphoglucomutase, phosphofructokinase, aldolase, PPi-fructose-6-P-1 phosphotransferase, and fructose-l,6-P₂ase, though predominantly cytosolic, were also present in the amyloplast. Based on distribution of enzymes, a probable pathway for starch biosynthesis in amyloplasts of developing wheat grains has been proposed.     

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.     

Characterization of ploidy levels of wheat microspore-derived plants using laser flow cytometry

Summary The purpose of this study was to determine simply and accurately ploidy levels as estimated by changes in nuclear DNA content of wheat (Triticum aestivum L.) plants regenerated from microspore-derived embryos. Using flow cytometry, the nuclear DNA content of green (83) and albino (222) plants derived using anther culture of ‘Bobwhite’ and ‘Pavon 76’, and of their reciprocal F₁ hydrids was estimated. The average DNA concent of the Bobwhite and Pavon 76 standards was 32.46 and 31.28 per nucleus, respectively. Microspore-derived haploid (3X), doubled-haploid (6X), nanoploid (9X), and dodecaploid (12X) plants contained on average 15.44, 30.56, 45.57, and 60.27 pg of DNA, respectively, at a ratio of 1∶1.98∶2.99∶3.90. The frequency of haploids (43.6%) was similar to that of doubled haploids (43.0%), and much larger than the frequency of endopolyploids [nanoploid (1.3%) and dodecaploid (1.0%)] and various aneuploids (11.1%). In terms of genetic stability, green plants had less chromosomal variation than albino plants. The procedure is suitable for rapid determination of the ploidy levels of wheat microspore-derived plants. The knowledge about DNA content or genome size of plants obtained here provides useful information to plant breeders and geneticists interested in using anther culture. Formerly of the Department of Agronomy, University of Nebraska, Lincoln. NE 68583-0915. Formerly of the Center for Biotechnology, University of Nebraska, Lincoln, NE 68588.     

Genetic aspects of wheat gliadin proteins

Inheritance of gliadin components unique to three different varieties of common wheat (Triticum aestivum L.) was studied in F₁ and F₂ seeds of intervarietal crosses using protein patterns obtained by polyacrylamide gel electrophoresis in aluminum lactate buffer (pH 3.2). The patterns of F₁ seeds of the crosses Cheyenne × Justin and INIA 66R × Justin evidenced all the bands present in the patterns of the parents; band intensities reflected gene dosage levels dependent on whether the contributing parent was maternal or paternal in accordance with the triploid nature of endosperm tissue. Most of the gliadin components examined segregated in accordance with control by a single dominant gene, but in two instances single bands in the one-dimensional electrophoretic patterns segregated in the F₂ as expected if controlled by two genes. A method of two-dimensional electrophoresis was developed that resolved these apparently single bands into two components each, which could segregate independently. Linkage analysis provided evidence of codominant alleles and closely linked genes coding for gliadin protein components in both coupling and repulsion situations. The gliadin protein components seem to be coded for by clusters of genes located on chromosomes of homoeologous groups 1 and 6 in hexaploid wheats.Reference to a company or product name does not imply approval by the U.S. Department of Agriculture to the exclusion of others which may also be suitable.