Wheat protein inhibitors of α-amylase

The location in the seed, molecular properties and biological role of protein α-amylase inhibitors from wheat are discussed. Inhibition specificity of albumin inhibitors and structural features essential for interaction with inhibited amylases are also examined. The possible significance of these naturally occurring inhibitors in relation to their presence in foods in active form is described. Finally, genetic aspects of the albumin inhibitor production and the possibility of improving nutritional value and insect re     

Intestinal Absorbability of Wheat Allergens,Subunits of a Wheat α-Amylase Inhibitor,Expressed by Bacteria

Wheat CM2, CM3 and CM16 proteins are known as subunits of the tetrameric α-amylase inhibitor as well as major allergens to baker’s asthma. The purpose of this study is to produce these CM proteins by bacteria in a quantity adequate for studying thepenetration characteristics of the CM proteins through intestinal mcosa in rats and Caco-2 cells. cDNAs encoding the mature proteins were expressed in Escherichia coli and purified by an Ni²⁺-chelating column. The recombinant proteins were radioiodinated and admministrered orally to rats or applied to the apical site of the Caco-2 cell monolayer. The radioactivity in the trichloroacetic acid-insoluble fraction, which was mainly composed of peptides with molecular mass less than that of the intact CM proteins, in the serum and the basolateral medium was highest in recombinant CM3. Accordingly, the intestinal absorption of these three proteins in the form present in wheat should be evaluated.     

Transgene × Environment Interactions in Genetically Modified Wheat

Background

The introduction of transgenes into plants may cause unintended phenotypic effects which could have an impact on the plant itself and the environment. Little is published in the scientific literature about the interrelation of environmental factors and possible unintended effects in genetically modified (GM) plants.

Methods and Findings

We studied transgenic bread wheat Triticum aestivum lines expressing the wheat Pm3b gene against the fungus powdery mildew Blumeria graminis f.sp. tritici. Four independent offspring pairs, each consisting of a GM line and its corresponding non-GM control line, were grown under different soil nutrient conditions and with and without fungicide treatment in the glasshouse. Furthermore, we performed a field experiment with a similar design to validate our glasshouse results.The transgene increased the resistance to powdery mildew in all environments. However, GM plants reacted sensitive to fungicide spraying in the glasshouse. Without fungicide treatment, in the glasshouse GM lines had increased vegetative biomass and seed number and a twofold yield compared with control lines. In the field these results were reversed. Fertilization generally increased GM/control differences in the glasshouse but not in the field. Two of four GM lines showed up to 56% yield reduction and a 40-fold increase of infection with ergot disease Claviceps purpurea compared with their control lines in the field experiment; one GM line was very similar to its control.

Conclusions

Our results demonstrate that, depending on the insertion event, a particular transgene can have large effects on the entire phenotype of a plant and that these effects can sometimes be reversed when plants are moved from the glasshouse to the field. However, it remains unclear which mechanisms underlie these effects and how they may affect concepts in molecular plant breeding and plant evolutionary ecology.     

Factors Affecting the Radiosensitivity of Hexaploid Wheat to γ-Irradiation: Radiosensitivity of Hexaploid Wheat (Triticum aestivum L.)

Understanding the radiosensitivity of plants, an important factor in crop mutation breeding programs, requires a thorough investigation of the factors that contribute to this trait. In this study, we used the highly radiosensitive wheat (Triticum aestivum L.) variety HY1 and J411, a γ-irradiation-insensitive control, which were screened from a natural population, to examine the factors affecting radiosensitivity, including free radical content and total antioxidant capacity, as well as the expression of TaKu70 and TaKu80 (DNA repair-related genes) as measured by real-time PCR. We also investigated the alternative splicing of this gene in the wild-type wheat ecotype by sequence analysis. Free radical contents and total antioxidant capacity significantly increased upon exposure of HY1 wheat to γ-irradiation in a dose-dependent manner. By contrast, in J411, the free radical contents exhibited a similar trend, but the total antioxidant capacity exhibited a downward trend upon increasing γ-irradiation. Additionally, we detected dose-dependent increases in TaKu70 and TaKu80 expression levels in γ-irradiated HY1, while in J411, TaKu70 expression levels increased, followed by a decline. We also detected alternative splicing of TaKu70 mRNA, namely, intron retention, in HY1 but not in J411. Our findings indicate that γ-irradiation induces oxidative stress and DNA damage in hexaploid wheat, resulting in growth retardation of seedlings, and they suggest that TaKu70 may play a causal role in radiosensitivity in HY1. Further studies are required to exploit these factors to improve radiosensitivity in other wheat varieties.     

Inheritance and Sequence Homology Analysis of the Maize DNA Introgressed into the Wheat Doubled Haploid Plant Through Wheat×Maize Cross

A maize (Zea mays L.) genome-specific repeated DNA sequence (clone MR64) has been transferred into one DH line of wheat through wheat (Triticum persicum Vav. ex Zhuk.) and maize cross. In the present study by RFLP analysis the authors proved that this DNA sequence could stably transmit into DH3 plants, the next generation derived from DH2 self-crossing. A similarity search in all DNA databases using BLASTN program showed that the DNA sequence of MR64 had as high as 93% identity to PREM-2 and 79% to Opie-2 in nucleotides. Both PREM-2 and Opie-2 are known as retrotransposons in maize genome, suggesting that MR64 likely is the partial sequence of a maize retrotransposon. Therefore, the results indicate that some retrotransposon might involve the DNA introgression from maize to wheat genome through wide fertilization. Stable inheritance of this maize genome-specific retrotransposon-like DNA in the wheat genome opens up the possibility of using retrotransposon as a new tool for gene tagging, function analysis, and insertional mutagenesis in wheat genome.     

Morphological Variability of Wheat Powdery Mildew in the Context of Its Parasitic Adaptation to Wheat–Aegilops Lines with Different Resistance

The interaction between the pathogen and wheat–Aegilops lines with different resistance as well as their parental forms in the course of powdery mildew infection was studied using scanning electron microscopy. The course of infection in the line 51/99i and its parental form, the Rodina variety, proved to be similar. The plants of both genotypes featured pronounced adhesion of the primary infection structures to the surface of plant epidermal cells and the formation of large, well-developed colonies of the fungus, which was evidence for parasite–host compatibility. The development of powdery mildew on the line 135/99i was similar to that on the parental form Aegilops speltoides K-389. The pathogen–host interaction was characterized by a longer incubation period. Sparse fungal colonies were formed from mycelial hyphae with multiple hyphal lobes, and their adhesion to the surface of the epidermal cells was disturbed in most cases. Such a pattern of pathogen development indicated that the host plants had some resistance factors operating mainly at the level of pathogen penetration. The types of resistance in lines 95/99i and 56/99i were not characteristic of the parental form Ae. speltoides K-389, but they were described for Ae. speltoides samples from other natural ranges (Ryabchenko et al., 2002). This fact suggests that the immune potential of Ae. speltoides as a species is polygenic, and its elements can be transmitted to hybrids irrespective of concrete plants used as the donors of resistance.     

New Variation Identified by SSR in a Wheat Variety Derived from Synthetic Hexaploid Wheat ( Triticum durum× Aegilops tauschii)

Hybridization and polyploidization are important ways for wheat to evolve and to genetically differentiate. Ninety two simple sequence repeat (SSR) molecular markers, which distributed in A, B, and D genomes , were used to perform genetic comparison between Chuan-W5436 (CW5436), a new wheat variety, and its parents, synthetic hexaploid wheat Syn786 (♀ ) and common wheat Mianyang 26 (My26) (♂). The results indicated that alleles were not genetically transmitted from parents (Syn786 (♀) crossed (My26) (♂) ) to the progeny CW5436 as Mendelian proportions . A new variation on a SSR molecular marker loci with novel additive bands was observed in CW5436 but not found in its parents. It suggested that artificial selective stress was an important factor to promote the frequency of significant deviations of the expected allele, resulting in microsatellite sequences of the progeny changed . The affect of the genetic differentiation of SSR molecular marker loci that occurred in wheat crosses and gene transfer on the genetic evolu1tion of wheat was discussed.     

The Effects of Water Stress on Photosystem Ⅱ in Wheat

The fluorescence yield at room temperature, the capacity of excitation energy distribution between photosystem Ⅰ and Ⅱ by Mg2+, variable fluorescence yield, variable fluorescence quenching rate and fluorescence complementary area were decreased under water stress. These indicated that photosystem Il was impaired. The inhibited variable fluorescence yield could be partly recovered by the addition of artificial electron donor DPC. Therefore, water stress inhibited not only the oxidizing site of photosystem Ⅱ but also impaired partly the reaction center of photosystem Ⅱ.     

Spring Wheat Leaf Appearance and Temperature: Extending the Paradigm?

Extensive research shows temperature to be the primary environmental factor controlling the phyllochron, or rate of leaf appearance, of wheat (Triticum aestivum L.). Experimental results suggest that soil temperature at crown depth, rather than air temperature above the canopy, would better predict wheat leaf appearance rates. To test this hypothesis, leaf appearance in spring wheat ('Nordic') was measured in a 2-year field experiment (Nunn clay loam soil; fine, smectitic, mesic Aridic, Argiustoll) with three planting dates and two soil temperature treatments. One temperature treatment (denoted +3C) consisted of heating the soil at crown depth to 3 degrees C above the ambient soil temperature (denoted +0C). Main stem cumulative leaf number was measured at least weekly until flag leaf emergence. Leaf appearance was essentially linear with both air and soil growing degree-days (GDD), although there was a stronger linear relationship with soil GDD in the +0C plants than in +3C plants. A weak positive relationship between planting date and the phyllochron was observed. Unexpectedly, we found that heating the soil did not increase the rate of leaf appearance, as the paradigm would predict. To explain these results, we propose extending the paradigm in two ways. First, three processes are involved in leaf appearance: (1) cell division at the shoot apex forms the primordium; (2) cell division in the intercalary meristem forms the cells that then (3) expand to produce the leaf. Cell division is predominantly controlled by temperature, but cell expansion is considerably more affected by factors other than temperature, explaining the influence of other factors on the phyllochron. Secondly, the vertical distribution of the two meristems and region of cell expansion occur over a significant distance, where temperature varies considerably, and temperature at a specific point (e.g. crown depth) does not account for the entire temperature regime under which leaves are developing.     

Water-Stress-Induced Ethylene Production in Wheat : A Fact or Artifact?

Effects of water stress on ethylene evolution from excised leaf segments and intact plants of wheat (Triticum aestivum L. cv Katepwa) were studied. Excised leaf segments of 8 day or 6 week old plants were dried until they lost 8% of their fresh weight (water potential about −2.3 megapascals). These and nondried control leaf segments (water potential about −1.0 megapascal) were sealed in glass tubes, and their ethylene production rates were compared by head space analysis via gas-chromatography. The dried leaves of both ages produced significantly more ethylene than the corresponding controls. However, when 6 week old intact plants were water-stressed by withholding water supply, and their ethylene production measured using a continuousflow system, no increase in ethylene was deteceted despite a drop in water potential to −2.9 megapascals over 6 days. Even the leaf segments excised from plants that had been subjected to water stress for 2, 4, or 6 days produced no more ethylene (in sealed tubes) than the leaves from well-watered plants. In fact, the ethylene production by these segments decreased with the increase in the severity of stress experienced by the plants. The results show that the commonly reported overproduction of ethylene by excised leaves subjected to rapid drying represents an artifact, which has little relevance to the water stress responses of intact wheat plants.     

Light-Induced Biogenesis of Chlorophyll–Protein Complexes in Developing Wheat Thylakoids

It is shown that the apoproteins of core complexes (CC) I and II, the - and -subunits of CF₁ ATP-synthase complexes, are present in seedlings grown under intermittent light (IML). The levels of light-harvesting complex (LHC) apoproteins in the 30- to 18-kD region increase rapidly upon exposure to continuous light (CL). The newly synthesized LHC apoproteins appear to be present predominantly in monomeric forms that later assemble into higher-order oligomeric forms. During the early stages of greening of wheat seedlings, polypeptides in the 20.5-19 and 17.5-15.5 kD regions (so-called early light-induced proteins (ELIP)) are observed, but they disappear fully after 6 h. As greening proceeds, the 727-nm band in low-temperature fluorescence spectra (77 K) gradually shifts to longer wavelength (740-742 nm), which clearly demonstrates the light-driven biogenesis of LHC I and its assembly with CC I.     

In Vitro Culture of Wheat and Genetic Transformation — Retrospect and Prospect

This review focuses on the improvement of wheat (Triticum aestivum L.) via tissue and cell culture and its use in gene transfer techniques. Success of the latter critically depends on the ability to regenerate plants from cells or tissues cultured in vitro. Hence, we have devoted attention to the attempts made so far in obtaining regenerants from diverse explants. Although it is known that immature embryos are the best source for initiating morphogenic cultures, basic information related to the process of differentiation can also be gained by studying less responding tissues. The opportunity provided by anther and microspore culture in wheat improvement and the progress made is also presented.

To enhance tissue culture responses, identification of chromosomes, gene loci, and genes is of cardinal importance. We have also surveyed the progress made in this regard by conventional but incisive plant-breeding techniques. Gene rearrangements in tissue culture leading to the appearance of somaclonal or gametoclonal variation are of interest in selection of useful cell lines. The last part of the review is devoted to work done on transient gene expression and transformation with emphasis on recent developments.     

Electrophoretic Survey of Proteins and Esterase Isozyme in Wheat×Maize Progenies

The analysis of soluble proteins and esterase isozyme in F2 progeny grains from wheat (Triticum aestivum L. ) × maize (Zea mays L. ) crosses indicated that the electrophoretic pattern of proteins and esterase isozymes was extremely different from that of their parents. Protein variation was mainly concentrated in the high-molecular-weight-Glu (HMW-Glu) zone. There were 5 kinds of protein eleetrophoretic patterns in the analyzed grains. VIZ: maternal, additional, complementary, hybrid and omission type which accounted for 22.6%, 14.3%, 15.5%, 30. 9% and 16.7% of the total tested grains respectively. In the analysis of esterase pattern, some variations in progenies were also found. The variations of electrophoretic pattern of proteins and esterase isozyme indicated that a genetic material change in wheat chromosomes could be induced in the distant hybridization.     

Wheat DNA polymerase CI: a homologue of rat DNA polymerase β

We have previously described a low-molecular-weight DNA polymerase (52 kDa) from wheat embryo: DNA polymerase CI (pol CI). This enzyme shares some biochemical properties with animal DNA polymerase (pol ). In this report, we analyse pol CI in wheat embryo germination. Immunodetection and measurement of the enzyme activity show that wheat pol CI remains at a constant level during germination, whereas dramatic changes of the replicative DNA polymerase A and B activities were previously reported. We observe that the level of pol CI in physiological conditions (embryo germination and dividing cell culture) is in agreement with a pol -type DNA polymerase. By microsequencing of the electroblotted 52 kDa polypeptide, we determined the sequence of a dodecapeptide from the N-terminal region. A comparative analysis of the N-terminal pol CI peptide with some mammalian pol sequences shows a clear homology with helix 1 of the N-terminal ssDNA domain (residues 15 to 26) of the rat pol . Thus, the helical structure of this region should be conserved in the wheat peptide. This represents the first evidence of a partial primary structure of a -type DNA polymerase in plants.     

Mechanisms of Meiotic Restitution and Their Genetic Regulation in Wheat–Rye Polyhaploids

Meiosis has been studied in partially fertile wheat–rye F₁ hybrids yielded by crosses Triticum aestivum (Saratovskaya 29 variety) × Secale cereale L. (Onokhoiskaya variety) (4x =28). Hybrid self-fertility proved to be caused by formation of restituted nuclei, which appear after equational segregation of univalent chromosome in AI and sister chromatid non-separation in AII of meiosis, as well as after AI blockage in three different ways. Both types of meiotic restitution were found in each hybrid plant. Expression of the meiotic restitution trait varied significantly in polyhaploids of the same genotype (ears of the same plants, anthers of the same ear, microsporocytes of the same anther). Chromatin condensation in prophase proved to be related to the division type and univalent segregation in AI. During reduction segregation of univalents in AI, sister chromatid cohesion and chromosome supercondensation remained unchanged. The results obtained suggest that in the remote hybrids with haploid karyotype of the parental origin (polyhaploids), the program of two-stage meiosis may be fundamentally transformed to ensure one instead of two divisions. We propose that meiotic restitution is a result of special genetic regulation of the kinetochore organization (both structural and functional) and chromatin condensation, i.e. of major meiotic mechanisms.     

Cloning and Sequence Analysis of cDNA 3′-end in Wheat DREB Gene

Adverse environmental conditions, such as drought and low temperature, greatly affect the plant‘s growth, development, survival and geographical distribution. They have become the critical factors which limit crop quality and productivity. To improve crop tolerance to these abiotic stresses,breeding researches have been focused on     

Properties and Specific Functional Features of Wheat Grain α-Amylase/Subtilisin Inhibitor

A protein bifunctional inhibitor of endogenous α-amylase and subtilisin has been isolated from wheat grain and purified. The inhibitor specifically inactivates α-amylase isozymes with high isoelectric point values (group α-AMY1) and has almost no effect on the α-AMY2 isozymes with low isoelectric point values. This enzyme does not belong to glycoproteins and has a molecular weight of 21 kDa and an isoelectric point of 7.2. The protein displays a relatively high thermostability and pH optimum of 8.0; its inhibitory activity requires the presence of Ca²⁺ cations. The inhibition of excess α-amylase in wheat grain with a low falling number by the purified protein is studied.