Identification of cytokinins in young wheat spikes (Triticum aestivum cv. Chinese spring)

Cytokinins in young wheat (Triticum aestivum cv. Chinese spring) spikes (2–15 mm) were isolated by immunoaffinity chromatography. High-performance liquid chromatography-radioimmunoassay and gas chromatography-mass spectrometry indicated that major cytokinins present weretrans-zeatin, ribosyl-trans-zeatin, ribosyldihydrozeatintrans-zeatin-9-glucoside, and the glucosides oftrans-zeatin, ribosyl-trans-zeatin, andtrans-zeatin-9-glucoside. Dihydrozeatin,iso-pentenyladenosine, andiso-pentenyladenine were also present but at lower concentrations.Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply approval to the exclusion of other products that may also be suitable. R. C. Durley's present address is Biological Sciences, Monsanto Company, St. Louis, Missouri 63167.     

Developmental Changes in Composition and Morphology of Cuticular Waxes on Leaves and Spikes of Glossy and Glaucous Wheat (Triticum aestivum L.)

The glossy varieties (A14 and Jing 2001) and glaucous varieties (Fanmai 5 and Shanken 99) of wheat (Triticum aestivum L.) were selected for evaluation of developmental changes in the composition and morphology of cuticular waxes on leaves and spikes. The results provide us with two different wax development patterns between leaf and spike. The general accumulation trend of the total wax load on leaf and spike surfaces is first to increase and then decrease during the development growth period, but these changes were caused by different compound classes between leaf and spike. Developmental changes of leaf waxes were mainly the result of variations in composition of alcohols and alkanes. In addition, diketones were the third important contributor to the leaf wax changes in the glaucous varieties. Alkanes and diketones were the two major compound classes that caused the developmental changes of spike waxes. For leaf waxes, β- and OH-β-diketones were first detected in flag leaves from 200-day-old plants, and the amounts of β- and OH-β-diketones were significantly higher in glaucous varieties compared with glossy varieties. In spike waxes, β-diketone existed in all varieties, but OH-β-diketone was detectable only in the glaucous varieties. Unexpectedly, the glaucous variety Fanmai 5 yielded large amounts of OH-β-diketone. There was a significant shift in the chain length distribution of alkanes between early stage leaf and flag leaf. Unlike C₂₈ alcohol being the dominant chain length in leaf waxes, the dominant alcohol chain length of spikes was C₂₄ or C₂₆ depending on varieties. Epicuticular wax crystals on wheat leaf and glume were comprised of platelets and tubules, and the crystal morphology changed constantly throughout plant growth, especially the abaxial leaf crystals. Moreover, our results suggested that platelets and tubules on glume surfaces could be formed rapidly within a few days.     

Chromosome banding in Triticum aestivum cv. chinese spring and Aegilops variabilis

N-banding has been used to identify each of the fourteen chromosomes of Aegilops variabilis Eig. These patterns differ from the patterns of the nine wheat chromosomes that band with this technique. The banding pattern of Aegilops variabilis chromosome G was the same when examined in the parent species and in the wheat-variabilis addition line. — Banding analysis can be reliably carried out on both mitotic and meiotic chromosomes of wheat. Nine pairs of wheat chromosomes can be identified at meiosis.     

Plant regeneration from protoplasts of Triticum aestivum L. cv. Nakasoushu

A fast-growing, small, granular, embryogenic callus was selected from primary calli induced from the Japanese wheat cultivar Nakasoushu and the Australian wheat cultivar Bodallin. Regenerable and fine suspension cultures were induced three to six months after liquid culture was initiated and were characterized by dense cytoplasm and active division. These suspension cultures routinely provided high yields of protoplasts with about 90% viability when incubated in a modified KMP (Kao and Michayluk, 1975) medium containing 1 mg l^-1 2,4-D (2,4-dichlorophenoxyacetic acid), and 1 mg l^-1 zeatin. Nakasoushu and Bodallin protoplasts divided at frequencies of 8.6% and 11.1%, respectively, in agarose-solidified media. When Nakasoushu protoplasts were cultured with effective nurse cells of sorghum and wheat, protoplast division increased to 16.9% and 12.6%, respectively. Plating efficiencies varied from 0.03% to 2.5%. After subculture, protocolonies yielded embryogenic calli and somatic embryos, from which green plants were eventually regenerated. Whole plants obtained from Nakasoushu protoplasts were fertile, demonstrating the first report of Japanese cultivars in wheat protoplast cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nucleolar organizers and fibrillar centres in Triticum aestivum L., cv. Chinese spring

Serial sectioning of wheat roots prepared for electron microscopy was used to count the number of fibrillar centres per nucleus and nucleolus and to calculate their sizes. After growth at 35 ° C for two days the nucleoli became segregated and a one to one relationship was evident between chromosomal nucleolar organizers and fibrillar centres. This was confirmed using an aneuploid line carrying an additional pair of organizers. Quantitative studies showed that the fibrillar centres occupied a volume 0.24% of the total chromatin reticulum. From this it was calculated that only about 1/3 of the fibrillar centre material was likely to be nucleolar organizer chromatin. The other material was considered to be the protein revealed in silver staining studies. The importance of this was shown by its constant ratio to the size of all nucleoli in a given nucleus. — Evidence was found for the movement and fusion of organizer flanking regions during growth at 35 ° C. The number of junctions between chromatin and nucleolar organizers drops by about half giving one per organizer after segregation, and serial sectioning demonstrated such junctions in close proximity, an arrangement suggestive of incipient fusion.     

Plant regeneration from protoplasts of wheat (Triticum aestivum cv. Hartog)

Morphologically normal green plants have reproducibly been regenerated from protoplasts of an Australian wheat (Triticum aestivum cv. Hartog). The protoplasts were isolated from fine embryogenic suspension cultures which were initiated from embryogenic callus. Protoplasts were incubated in a modified liquid MS medium containing half strength of the macroelements, 5 m 2,4-D and 0.6 M glucose. Colonies were formed at frequencies ranging from 0.1% to 5%. The frequency of colonies forming fully developed plants varied between 1% and 25%. More than eighty green plants with morphologically normal shoots and roots have been obtained and there was no difficulty in establishing these plants in soil. A cytological study of several randomly selected regenerated plants showed the normal chromosome complement for wheat (2n = 42).     

中国春双端二体中端体的配对研究

21个中国春双端二体与中国春品种杂交,观察了杂种F_1减数分裂中期Ⅰ端体与相应染色体的配对。结果表明:14个组合的中期Ⅰ具有1个异形三价体的PMC的百分数超过90%;有3个组合(6A、5B和6D)分别为89.87%、83.56%和85.71%;2个组合(4A和1B)低于80%。在4A、1B和5B的3个组合中,具有1个异形二价体和1个单价端体的PMC超过15%。在4A、1B和4D的组合中,还有一定频率的PMC具有2个单价端体。用21个组合中端体配对频率计算的二价体频率与中国春品种中期Ⅰ构型频率基本一致。这些结果表明,染色体臂作为端体时和它作为双臂染色体的一部分时,是以同样的方便程度配对的。     

Optical maps refine the bread wheat Triticum aestivum cv. Chinese Spring genome assembly

Until recently, achieving a reference-quality genome sequence for bread wheat was long thought beyond the limits of genome sequencing and assembly technology, primarily due to the large genome size and > 80% repetitive sequence content. The release of the chromosome scale 14.5-Gb IWGSC RefSeq v1.0 genome sequence of bread wheat cv. Chinese Spring (CS) was, therefore, a milestone. Here, we used a direct label and stain (DLS) optical map of the CS genome together with a prior nick, label, repair and stain (NLRS) optical map, and sequence contigs assembled with Pacific Biosciences long reads, to refine the v1.0 assembly. Inconsistencies between the sequence and maps were reconciled and gaps were closed. Gap filling and anchoring of 279 unplaced scaffolds increased the total length of pseudomolecules by 168 Mb (excluding Ns). Positions and orientations were corrected for 233 and 354 scaffolds, respectively, representing 10% of the genome sequence. The accuracy of the remaining 90% of the assembly was validated. As a result of the increased contiguity, the numbers of transposable elements (TEs) and intact TEs have increased in IWGSC RefSeq v2.1 compared with v1.0. In total, 98% of the gene models identified in v1.0 were mapped onto this new assembly through development of a dedicated approach implemented in the MAGAAT pipeline. The numbers of high-confidence genes on pseudomolecules have increased from 105 319 to 105 534. The reconciled assembly enhances the utility of the sequence for genetic mapping, comparative genomics, gene annotation and isolation, and more general studies on the biology of wheat.     

Molecular cytogenetic analysis of DNA sequences with flanking telomeric repeats in Triticum aestivum cv. Begra.

A cloned genomic DNA fragment (pTa241) formerly derived from a DNA fraction obtained from isolated nuclei of embryos of a Polish cultivar of wheat (Triticum aestivum cv. Begra) comprises a tandem repeat of the telomeric array CCCTAAA, and hybridizes in situ exclusively to the telomeres of all chromosome arms of the somatic chromosome complement of wheat. A second cloned fragment (pTa637) derived from the same fraction is 637 bp long, flanked by 28 bp of the same telomeric repeat unit, and hybridizes in situ to the entire lengths of all the chromosomes of the complement. The same pattern of hybridization was observed when the flanking telomeric sequences were removed. A third DNA fragment (pTa1439), derived from unfractionated genomic DNA and flanked with 62 bp of the same telomeric unit, showed the same patterns of distribution. Together with additional evidence from Southern analysis, these observations were interpreted to mean that these sequences are associated with mobile DNA elements and are distributed widely throughout the genome. The chromosomal distribution of the non-telomeric parts of the clones is consistent with the dispersed genomic distribution characteristic of transposons and retroelements.     

Characterization of a cysteine protease from wheat Triticum aestivum (cv. Giza 164)

Enzymes, especially proteases, have become an important and indispensable part of the processes used by the modern food and feed industry to produce a large and diversified range of products for human and animal consumption. A cysteine protease, used extensively in the food industry, was purified from germinated wheat Triticum aestivum (cv. Giza 164) grains through a simple reproducible method consisting of extraction, ion exchange chromatography and gel filtration. The molecular weight of the enzyme was estimated to be 61000+/-1200-62000+/-1500 by SDS-PAGE and gel filtration. The cysteine protease had an isoelectric point and pH optimum at 4.4 and 4.0, respectively. The enzyme exhibited more activity toward azocasein than the other examined substrates with K(m) 2.8+/-0.15 mg azocasein/ml. In addition, it had a temperature optimum of 50 degrees C and based on a heat stability study 55% of its initial activity remained after preincubation of the enzyme at 50 degrees C for 30 min prior to substrate addition. All the examined metal cations inhibited the enzyme except Co(2+), Mg(2+), Mn(2+) and Li(+). The proteolytic activity of the enzyme was inhibited by thiol-specific inhibitors, whereas iodoacetate and p-hydroxymercuribenzoate caused a competitive inhibition with Ki values 6+/-0.3 mM and 21+/-1.2 microM, respectively. Soybean trypsin inhibitor had no effect on the enzyme. The enzyme activity remained almost constant for 150 days of storage at -20 degrees C. The properties of this enzyme, temperature and pH optima, substrate specificity, stability and sensitivity to inhibitors or activators, meet the prerequisites needed for food industries.     

Molecular and biochemical characterization of cytosolic phosphoglucomutase in wheat endosperm (Triticum aestivum L. cv. Axona)

Evidence from a number of plant tissues suggests that phosphoglucomutase (PGM) is present in both the cytosol and the plastid. The cytosolic and plastidic isoforms of PGM have been partially purified from wheat endosperm (Triticum aestivum L. cv. Axona). Both isoforms required glucose 1,6-bisphosphate for their activity with K(a) values of 4.5 micro M and 3.8 micro M for cytosolic and plastidic isoforms, respectively, and followed normal Michaelis-Menten kinetics with glucose 1-phosphate as the substrate with K(m) values of 0.1 mM and 0.12 mM for the cytosolic and plastidic isoforms, respectively. A cDNA clone was isolated from wheat endosperm that encodes the cytosolic isoform of PGM. The deduced amino acid sequence shows significant homology to PGMs from eukaryotic and prokaryotic sources. PGM activity was measured in whole cell extracts and in amyloplasts isolated during the development of wheat endosperm. Results indicate an approximate 80% reduction in measurable activity of plastidial and cytosolic PGM between 8 d and 30 d post-anthesis. Northern analysis showed a reduction in cytosolic PGM mRNA accumulation during the same period of development. The implications of the changes in PGM activity during the synthesis of starch in developing endosperm are discussed.     

Identification and verification of microRNA in wheat (Triticum aestivum)

MicroRNAs (miRNAs) are small, endogenous RNAs that regulate gene expression in both plants and animals. A large number of miRNAs has been identified from various animals and model plant species such as Arabidopsis thaliana and rice (Oryza sativa); however, characteristics of wheat (Triticum aestivum) miRNAs are poorly understood. Here, computational identification of miRNAs from wheat EST sequences was preformed by using the in-house program GenomicSVM, a prediction model for miRNAs. This study resulted in the discovery of 79 miRNA candidates. Nine out of 22 miRNA representatives randomly selected from the 79 candidates were experimentally validated with Northern blotting, indicating that prediction accuracy is about 40%. For the 9 validated miRNAs, 59 wheat ESTs were predicted as their putative targets. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Weibo Jin and Nannan Li contributed equally to the work.     

cDNA encoding a wheat (Triticum aestivum cv. Chinese Spring) glycine-rich RNA-binding protein

A wheat cDNA encoding a glycine-rich RNA-binding protein, whGRP-1, was isolated. WhGRP-1 contains two conserved domains, the RNA-binding motif (RNP motif) combined with a series of glycine-rich imperfect repeats, characteristic of a conserved family of plant RNA-binding proteins. Northern analysis revealed that whGRP-1 mRNA accumulates to high levels in roots and to lower levels in leaves of wheat seedlings. whGRP-1 mRNA accumulation is not enhanced by exogenous abscisic acid in seedlings and accumulates to very high levels during wheat embryo development, showing a pattern different from that of the ABA-inducible wheat Em gene.     

Identification of C-banded chromosomes in meiosis of common wheat,Triticum aestivum L.

Summary The C-banding pattern of nine meiotic chromosomes of common wheat (Triticum aestivum L.) as described. In F₁s of crosses between monosomics of Chinese Spring and two Spanish wheat cultivars, univalent chromosomes were used to aid the recognition and analysis of the C-banding pattern for the individual chromosomes. The identification of one chromosome involved in one translocation in Chinese Spring x Pané 247 has been made through heterochromatin bands observed in the chromosomes involved in multivalents.     

Production of near-isogenic lines and marked monosomic lines in common wheat (Triticum aestivum) cv. Chinese Spring.

Sixteen near-isogenic lines (NILs) carrying a marker gene were produced by the recurrent backcrossing method in the genetic background of common wheat (Triticum aestivum) cv. Chinese Spring (CS). Three genes from alien species showed segregation distortion. In NILs carrying a marker gene of rye (Secale cereale) or Aegilops caudata, the alien chromosome segments were detected by fluorescence in situ hybridization (FISH). The NILs were grown with replications and the effect of marker genes on plant morphology in the genetic background of CS was investigated. These NILs were further crossed with the corresponding monosomics of CS and 13 monosomic lines whose monosome carries a respective marker gene were established and named "marked monosomics." Many of the marked monosomics were distinguishable from the disomic NILs because of the different dosage effect of the genes. The NILs are utilized for studies on gene isolation or gene regulation. Marked monosomics are useful not only for monosomic analysis but also for production of homologous chromosome substitution lines because chromosome observation is not required.     

Cytological and anatomical observations on the awn and lemma of wheat (Triticum aestivum L. cv. Ofanto)

Abstract

The anatomy and cytology of the awn and lemma of Triticum aestivum cv. Ofanto was studied. Transverse sections of awns showed five vascular bundles, elongated and branched chlorenchyma cells containing protein bodies are lacking in starch; therefore sugars are supposed rapidly translocated. Starch is abundant in the spike. Phytoliths are present.     

137Cs uptake in spring wheat (Triticum aestivum L.cv. Tonic) at varying K supply

Spring wheat (Triticum aestivum L. cv. Tonic) was grown for 16 days in a sandy loam soil which was contaminated with ¹³⁷Cs. The soil was fertilised with K at three rates (0,1 and 2 mmol K per 950 g dry soil) and with NO₃ ^--N at two rates (0 and 2 mmol per 950 g dry soil) in a factorial design. The ¹³⁷Cs Activity Concentration (AC) in the shoot tissue significantly reduced 8.2-fold (nil N treatment, p<0.001) and 9.3-fold (highest N dose, p<0.001) with increasing K supply. In contrast, the K application increased the ¹³⁷Cs AC in soil solution 1.7 fold (nil N treatment) or had no significant effect (highest N dose). At similar K application, the application of N increased the ¹³⁷Cs AC in the shoot compared to the control. This effect is most probably due to the increased NH₄ ⁺ concentration in soil solution which increased the ¹³⁷Cs AC in soil solution. The soil solution composition (¹³⁷Cs and K concentration) in the rhizosphere was estimated from the average soil solution composition at day 16 and solute transport calculations. The ¹³⁷Cs AC in the shoot tissue was predicted from the estimated soil solution composition in the rhizosphere and the relationship between K concentration and ¹³⁷Cs uptake derived from a nutrient solution experiment. The predictions of ¹³⁷Cs AC's in the shoot are qualitatively correct for the fertiliser effects but underestimate the observations between 1.4 and 9.9 fold.     

Molecular cloning and characterization of an up-regulated UDP-glucosyltransferase gene induced by DON from Triticum aestivum L. cv. Wangshuibai

Fusarium head blight, also called scab, is a serious disease of small grain cereals and maize. Scab can not only cause yield loss, more seriously is that it can also deteriorate seed quality by contaminating the infected grains with trichothecenes toxins harmful to human and animal health. Deoxynivalenol (DON) is one of the most important toxin members. It was proposed that DON acted first as a virulence factor during fungal pathogenesis and then accumulated in grain to levels posing a threat to human and animal health. In the present research, by expression analysis of DON-induced samples using GeneChip^® Wheat Genome Array (http://www.affymetrix.com/products/arrays/specific/wheat.affx), a DON-resistance related gene TaUGT3 (GenBank accession FJ236328) was cloned and characterized from a scab resistant wheat (Triticum aestivum L.) variety Wangshuibai. The full-length cDNA of TaUGT3 was 1,755 bp and contained a putative open reading frame (ORF) with 496 amino acids encoding a UDP-glucosyltransferase (UGT). TaUGT3 showed high similarity in amino acid level with DOGT1 gene in Arabidopsis, which is able to detoxify DON. TaUGT3 was located on the group 3 chromosomes of wheat using nulli-tetrasomic lines and deletion lines of Chinese Spring. Co-transformed of TaUGT3 with GFP genes to onion epidermis cells using transient transformation technique by microprojectile bombardment indicated the subcellular location of the protein encoded by TaUGT3 was in the plasma membrane and nuclear. Transformation and overexpression of the TaUGT3 gene in Arabidopsis could enhance tolerance against DON.