Chromatid and Chromosome Type Breakage-Fusion-Bridge Cycles in Wheat (Triticum Aestivum L.)

During the development of disomic additions of rye (Secale cereale L.) chromosomes to wheat (Triticum aestivum L.), two reverse tandem duplications on wheat chromosomes 3D and 4A were isolated. By virtue of their meiotic pairing, the reverse tandem duplications initiated the chromatid type of the breakage-fusion-bridge (BFB) cycle. This BFB cycle continued through pollen mitoses and in the early endosperm divisions, but no clear evidence of its presence in embryo mitoses was found. The chromosome type of BFB cycle was initiated by fusion of two broken chromosome ends resulting in a dicentric or a ring chromosome. Chromosome type BFB cycles were detected in embryo mitoses and in root tips, but they did not persist until the next meiosis and were not transmitted to the progeny. Active BFB cycles induced breakage of other wheat chromosomes that resulted in additional reverse tandem duplications and dicentric and ring chromosomes. Four loci, on chromosome arms 2BS, 3DS, 4AL, and most likely on 7DL, were particularly susceptible to breakage. The BFB cycles produced high frequency of variegation for pigmentation of the aleurone layer of kernels and somatic chimeras for a morphological marker. With the exception of low mutation rate, the observed phenomena are consistent with the activity of a Ds-like element. However, it is not clear whether such an element, if indeed present, was of wheat or rye origin.     

Purification of TAXI-like Endoxylanase Inhibitors from Wheat (Triticum Aestivum L.) Whole Meal Reveals a Family of Iso-forms

An affinity chromatography method has been developed for purification of endoxylanase inhibitors concentrated by cation exchange chromatography from wheat whole meal and is based on immobilisation of a Bacillus subtilis family 11 endoxylanase on N -hydroxysuccinimide activated Sepharose 4 Fast Flow. When followed by high-resolution cation exchange chromatography, the purification of seven TAXIs, Triticum aestivum L. endoxylanase inhibitors was achieved so extending the number of such proteins known to date (TAXI I and II). Based on their inhibition activities against a B. subtilis family 11 and an Aspergillus niger family 11 endoxylanase, six TAXI I- and only one TAXI II-like inhibitor could be distinguished. The first type of endoxylanase inhibitor is active against both endoxylanases and the second type only has significant activity against the B. subtilis endoxylanase.     

Purification and Characterization of Lipase from Wheat (Triticum aestivum L.) Germ

A method of isolation and purification of lipase (EC 3.1.1.3) from the germ of wheat (Triticum aestivumL.) is described. An electrophoretically homogeneous preparation of the enzyme (specific activity, 622.5 × 10^–3 mol/min per mg protein) was obtained after 61-fold purification. The molecular weight of the enzyme, determined by gel chromatography, was 143 ± 2 kDa. The optimal conditions for the enzyme were 37°C and pH 8.0. The homogeneous preparation of the lipase exhibited high thermal stability: over 20% of the original activity was retained after incubation of the preparation at high temperatures (60–90°C) for 1 h at pH 8.0.     

小麦TaPSG719基因启动子的分离及序列分析

TaPSG719基因是从小麦中分离的花粉特异性表达基因,其功能未知.克隆和分析该基因的启动子有助于研究该基因的功能,解析小麦花器官的发育调控机制.本研究根据已报道的TaPSG719基因cDNA序列为基础设计引物,经过两次反向PCR获得了该基因起始密码子上游1776 bp的调控序列.应用PLACE和PlantCARE数据库系统对该序列进行分析研究,发现其具有启动子的基本元件TATA-box和CAAT-box、两种花粉特异性调控元件AGAAA和GTGA及光反应和激素响应元件.     

Arsenic Transformation by Azospirillum Brasilense Sp245 in Association with Wheat (Triticum Aestivum L.) Roots

The transformation of sodium arsenite and sodium arsenate by the rhizospheric nitrogen-fixing bacterium Azospirillum brasilense Sp245 in association with wheat (Triticum aestivum L. ‘Saratovskaya 29’) was studied. The effect produced by the A. brasilense strain on the morphological parameters of wheat in an As-polluted environment was examined. The plants were cultivated in a hydroponic system, with glass beads serving as a support for root growth. The plant-growth medium (an artificial soil solution) was deficient in P and Fe. The total initial As concentrations used were 75, 750, and 7500 μg l⁻¹. The As compounds used contained sodium arsenate and sodium arsenite at an As(V):As(III) ratio of 1:3.6 (in terms of As) in all experiments. Inoculation of A. brasilense Sp245 led to a decrease in the overall root length and to the formation of lateral roots; both effects are possibly related to the bacteria’s ability to synthesize auxins. Inoculation also changed the As(V): As(III) ratio of the plant-growth medium. In all experiments, the concentration of As(V) in the nutrient medium increased relative to the initial one and was approximately 1.5-fold higher than that in the medium of uninoculated plants. This value slightly decreased (1.6 > 1.5 > 1.4) with increasing concentration of As in the medium. Azospirillum-inoculated plants accumulated less As than did the surface-sterilized uninoculated plants. This study shows that A. brasilense Sp245 in association with wheat changes the speciation, bioavailability, and plant uptake of As.     

Partial Purification and Characterization of UDP-Glucose Pyrophosphorylase from Immature Grains of Wheat (Triticum aestivum L.)

Uridine diphosphate glucose pyrophosphorylase (UDP-Glc PPase, EC2.7.7.9) was purified 65 fold from immature grains of wheat (Triticum aestivum L. cv, WH-147) by ammonium sulphate fractionation, DEAE-cellulose anion exchange chromatography and Sephadex G-100 permeation chromatography. The partially purified enzyme, having molecular weight of 72 kD, exhibited broad pH optimum between 8 and 9 and was stable at 4°C for 15 days. At pH 8.5, the enzyme followed typical hyperbolic kinetics with respect to UDP-glucose and inorganic pyrophosphate (Km 0.22 mM and 0.66 mM respectively). The enzyme showed absolute requirement for Mg²⁺ and did not appear to require sulfhydryl groups for its activity. Initial velocity and product inhibition studies indicated sequential addition of substrates and sequential release of products.     

Purification and Characterization of an Iminopeptidase from the Primary Leaf of Wheat (Triticum aestivum L.)

Iminopeptidase (EC 3.4.11.5) was substantially purified from the primary leaves of 7-day-old wheat seedlings (Triticum aestivum L.). The purification procedure consisted of five steps: acid precipitation, molecular exclusion chromatography on Sephacryl S-200, Ultrogel AcA 44, Sepharose 2B and ion-exchange chromatography on DEAE-cellulose. Iminopeptidase isolated in this manner was only active against the β-naphthylamides of proline and hydroxyproline. For each substrate, the pH optimum was 7.4 and activity was sensitive to sulfhydryl group inhibitors. The iminopeptidase hydrolyzed the dipeptides Pro-Leu, Pro-Gly, Hyp-Gly, and Pro-Tyr. Iminopeptidase activity against the dipeptide Pro-Gly was higher than against Hyp-Gly. The molecular weight was estimated to be about 400,000. Evidence was obtained for the existence of endogenous inhibitors of iminopeptidase activity.     

Development and Characterization of a New TILLING Population of Common Bread Wheat (Triticum aestivum L.)

Mutagenesis is an important tool in crop improvement. However, the hexaploid genome of wheat (Triticum aestivum L.) presents problems in identifying desirable genetic changes based on phenotypic screening due to gene redundancy. TILLING (Targeting Induced Local Lesions IN Genomes), a powerful reverse genetic strategy that allows the detection of induced point mutations in individuals of the mutagenized populations, can address the major challenge of linking sequence information to the biological function of genes and can also identify novel variation for crop breeding. Wheat is especially well-suited for TILLING due to the high mutation densities tolerated by polyploids. However, only a few wheat TILLING populations are currently available in the world, which is far from satisfying the requirement of researchers and breeders in different growing environments. In addition, current TILLING screening protocols require costly fluorescence detection systems, limiting their use, especially in developing countries. We developed a new TILLING resource comprising 2610 M(2) mutants in a common wheat cultivar 'Jinmai 47'. Numerous phenotypes with altered morphological and agronomic traits were observed from the M(2) and M(3) lines in the field. To simplify the procedure and decrease costs, we use unlabeled primers and either non-denaturing polyacrylamide gels or agarose gels for mutation detection. The value of this new resource was tested using PCR with RAPD and Intron-spliced junction (ISJ) primers, and also TILLING in three selected candidate genes, in 300 and 512 mutant lines, revealing high mutation densities of 1/34 kb by RAPD/ISJ analysis and 1/47 kb by TILLING. In total, 31 novel alleles were identified in the 3 targeted genes and confirmed by sequencing. The results indicate that this mutant population represents a useful resource for the wheat research community. We hope that the use of this reverse genetics resource will provide novel allelic diversity for wheat improvement and functional genomics.     

小麦高分子量麦谷蛋白亚基1Dx5启动子驱动外源基因的表达

将小麦高分子量麦谷蛋白亚基(HMW-GS)基因的胚乳组织特异性表达启动子驱动的外源突变型1Dx5基因和gus基因导入小麦中.对其转基因植株连续3代的跟踪研究表明,突变型1Dx5基因的重复序列导致其表达蛋白分子量增大,并影响其它1Bx17 1By18亚基基因的表达.组织化学分析观察到gus基因在1Dx5基因启动子驱动下的表达表现出胚乳组织特异性,在开花2周后开始表达,表达量呈持续上升,至腊熟期达到最高,其次为籽粒成熟期.     

Free Polyamines in Senescing Wheat(Triticum aestivum L.)

The free polyamine content of flag leaves, peduncles, rachis,glumes, and grains of wheat (Triticum aestivum L., cv. Castell)plants, ripening under field conditions, has been investigatedduring three consecutive growing seasons. Putrescine was quantitativelythe most important of all polyamines detected in these organs.Concentrations were highest in the grains, glumes and flag leaves.No correlation was found between polyamine content and the onsetof senescence of flag leaves and other organs. Excised primaryleaves, however, showed a decrease in polyamine content in thedark and also in light/dark cycles, but in the latter case onlyafter an initial increase. Sink removal of otherwise intactwheat plants caused an accumulation of putrescine in flag leavesat the later stages of senescence, whereas removal of all otherleaves was without any significant effect. Putrescine was alsorecovered in phloem-exudate samples collected throughout theperiod of grain development. In both grains and glumes, peakconcentrations of polyamines were found early during seed development. Key words: Triticum aestivum, polyamines, ripening, senescence     

Regeneration of Freezing-Tolerant Spring Wheat (Triticum aestivum L.) Plants from Cryoselected Callus

A cryoselection protocol has been developed that provides freezing-tolerant callus that, in turn, can regenerate plants with enhanced cold hardiness. Tolerant calli were selected from spring wheat (Triticum aestivum L.) callus by immersion in liquid nitrogen without addition of cryoprotectants. Less than 15% of the calli survived the initial challenge, whereas 30 to 40% of previously selected calli survived subsequent exposure. Seed progeny from five of 11 regenerant (R2) lines tested exhibited significantly enhanced tolerance to freezing at −12°C. Thus, cryoselection appears to involve at least in part, selection for genetic rather than epigenetic variants. Analysis of one callus line indicated that cryoselection did not induce significant alterations in lipid composition, adenylate energy charge, or freezing point. An increase in the soluble sugar component was detected. Changes were also detected in the protein complement of microsomal membrane and soluble protein extracts of cryoselected callus. In all, seven unique proteins ranging from 79 to 149 kilodaltons were identified. The results demonstrate that freezing tolerant callus can be isolated from a heterogeneous population by cryoselection, and factors that contribute to hardiness at the callus level are biologically stable and can contribute to tolerance at the whole plant level.     

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.     

Callus Formation from Somatic Hybridization of Wheat (Triticum aestivum L.) and Ryegrass (Lolium perenne L.) by Electrofusion

Somatic hybrid calli were recovered following electrofusion of protoplasts from a chloroplast-containing cell suspension culture of wheat (Triticum aestivum L.) and a cell suspension culture of ryegrass (Lolium perenne L.). The protoplasts of wheat were inactivated by iodoacetamide; in addition morphology and colour were used as markers to aid selection of putative hybrid calli. For isozyme analysis of putative hybrids, nine isozymes were tested for differences in bands between the parental lines. Of these, three showed differences (ADH, GOT, SDH). Analysis of ADH bands of calli indicated that six lines were hybrids. These lines were analysed with the ,ther isozymes, and at the DNA level by Southern hybridisation with a wheat ribosomal DNA probe. The overall results indicated that one line was an almost complete combination of the genomes of the parental lines, but the other 5 lines were probably partial hybrids. In the latter, some loss of the wheat genome had probably occurred.     

Light-dependent Proton Excretion of Wheat (Triticum aestivum L.) and Maize (Zea mays L.) Roots

We investigated the change of root net proton excretion of seedlings of Triticum aestivum L. and Zea mays L. with daily variation of illumination using a multi-channel pH-stat system. We found an increase of net proton excretion during darkness and a drop after the beginning of illumination. Inhibition of carotenoid biosynthesis by norflurazone and photooxidation of chlorophylls did not change the periodicity or its induction. The induction of diurnal periodicity was possible with blue, green and red light. After induction the oscillation of net proton excretion continued for at least two cycles under constant light. We conclude that net H⁺ excretion of wheat and maize roots may be regulated by an endogenous clock or by a signal from the leaves. The nature of such a hypothetical signal remains unknown.     

Properties of ATP-dependent Phosphofructokinase from Endosperm of Developing Wheat (Triticum aestivum L.) Grains

ATP-dependent phosphofructokinase (ATP:D-fructose S-phosphate, 1-phosphotransferase, EC 2.7.1.11, PFK) from endosperm of developing wheat grains was purified to apparent homogeneity with about 45% recovery using ammonium sulfate fractionation, ion-exchange chromatography on DEAE-cellulose and gel filtration through Sepharose CL-SB and Sephadex G-200. The purified enzyme with a molecular weight of about 182 kD, was a heterotetramer with subunit molecular weights ranging between 20 and 80 kD. The enzyme exhibited maximum activity at pH 7.9 and was highly specific for its substrates. The enzyme had absolute requirement for Mg2+. At pH 7.9, the Km values as determined by Lineweaver-Burk plots were 1.43 and 0.70 mM, respectively for fru-S-P and ATP. Fru-2, S-P2 had no effect on the activity of the enzyme. The enzyme was inhibited strongly by citrate, ADP, 3-PGA and PEP with Ki values of 2.40, 1.75, 2.10 and 0.80 mM, respectively. Citrate and PEP inhibited the enzyme competitively with respect to both fru-S-P and ATP. ADP and 3-PGA inhibited the enzyme non-competitively and competitively, respectively with respect to fru-S-P and in a mixed manner with respect to ATP. Hill plot values indicated co-operative interaction of citrate, 3-PGA and PEP with the enzyme.     

Characterization of amyloplastic phosphohexose isomerase from immature wheat (Triticum aestivum L.) endosperm

Phosphohexose isomerase from amyloplasts of immature wheat endosperm was purified 133-fold. The enzyme had a molecular weight of 130 kDa and maximum activity at pH 8.6. It showed normal hyperbolic kinetics for both fructose-6-P and glucose-6-P with Km of 0.12 mM and 0.44 mM, respectively. pH had a great influence on Km for fructose-6-P. Using glucose-6-P as the substrate, the equilibrium was reached at 23% fructose-6-P and 77% glucose-6-P and an equilibrium constant of about 3.0. The delta F calculated from the apparent equilibrium constant was +742 cal.mol-1. The activation energy calculated from the Arrhenius plot was 7450 cal.mol-1. None of the sulphydryl reagents at 2.5 mM concentration inactivated the enzyme. The enzyme was competitively inhibited by 6-phosphogluconate, ribose-5-P and ribulose-5-P with Ki values of 0.18, 0.14, and 0.13 mM, respectively. The probable role of the enzyme in starch biosynthesis in amyloplasts is discussed.     

Colchicine-induced Paracrystals in Root Cells of Wheat (Triticum aestivum L.)

Tubulin conformations other than microtubules in the meristematiccells of wheat roots grown in the presence of 2 mM colchicinesolution were investigated by immunofluorescence and electronmicroscopy. In the affected cells microtubules disappeared andwere replaced by tubulin fluorescent strands that occurred inthe cortical cytoplasm. With increasing time of exposure tocolchicine the tubulin strands became better organized and occurredalso in the subcortical cytoplasm and finally they were restrictedto the area around the nucleus. In prophase and preprophasecells thick strands occupied the cortical cytoplasmic zone wherein normal cells a preprophase microtubule band (PMB) was expectedto be assembled. In the colchicine-treated cells electron microscopy revealedan accumulation of paracrystalline aggregates, which initiallyoccurred along the cell wall and later deeper in the cytoplasm,in the perinuclear regions and the cytoplasmic invaginationsof the nucleus. In transverse planes the paracrystalline strandsappear to consist of hexagonal subunits in a 'honeycomb' arrangement,while in longitudinal and oblique sections they exhibit variableimages. Since their distribution coincides with that of thetubulin strands visualized by immunofluorescence, they are consideredto be the same structure. Therefore, the paracrystals consistof, or at least contain, tubulin. They are most likely to bepolymers of tubulin-colchicine complexes.Copyright 1995, 1999Academic Press Wheat roots, colchicine, immunofluorescence, electron microscopy, tubulin paracrystals, Triticum aestivum L     

小麦内切多聚半乳糖醛酸酶抑制蛋白的分离纯化研究

小麦内切多聚半乳糖醛酸酶抑制蛋白的分离纯化研究郑远旗,杨宗剑,李建吾,周立,吴文莲（四川大学生物系,成都６１００６４）余露（中国科学院成都生物研究所,成都６１００４１）关键词内切多聚半乳糖醛酸酶抑制蛋白;内切多聚半乳糖醛酸酶;纯化;小麦内切多聚半乳糖...     

Localization of Galactolipid Biosynthesis in Etioplasts Isolated from Dark-Grown Wheat (Triticum aestivum L.)

Etioplasts were isolated from leaves of dark-grown wheat (Triticum aestivum L. var Starke II). Galactolipid biosynthesis was assayed in an envelope-rich fraction and in the fraction containing the rest of the etioplast membranes by measuring incorporation of ¹⁴C from uridine-diphospho[¹⁴C]galactose into monogalactosyl diacylglycerol and digalactosyl diacylglycerol. More than half of the galactolipid biosynthetic capability was found in the fraction of inner etioplast membranes. This fraction was subfractioned into fractions enriched in prolamellar bodies and membrane vesicles (prothylakoids), respectively. All membrane fractions obtained from etioplasts were able to carry out galactolipid biosynthesis, although the activity was very low in prolamellar body-enriched fractions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed markedly different polypeptide patterns between the different fractions. It is concluded that the capability of galactolipid biosynthesis of etioplasts probably is not restricted to the envelope, but is also present in the inner membranes of this plastid.     

Characterization of a Putative New Semi-Dominant Reduced Height Gene,Rht_NM9, in Wheat(Triticum aestivum L.)

Plant height is an important agronomic trait in cereal crops, and can affect both plant architecture and grain yield. New dwarfing genes are required for improving the genetic diversity of wheat. In this study, a novel dwarf mutant, NM9, was created by treating seeds of the wheat variety NAU9918 with ethyl methanesulfonate(EMS). NM9 showed obvious phenotypic changes, which were distinct from those caused by other dwarfing genes, especially the reduced plant height, increased effective tiller number, and elongated spike and grain length. The reduced plant height in NM9 was attributable to a semi-dominant dwarfing gene Rht_NM9, which was flanked by two closely linked SNP markers, SNP34 and SNP41, covering an 8.86-Mb region on the chromosome arm 2AS. The results of gibberellic acid(GA) sensitivity evaluation, comparative genomics analysis and allelism test indicated that Rht_NM9 was neither allelic to Rht7 and Rht21 nor homoeoallelic to Rht8, so Rht_NM9 was proposed to be a new dwarfing locus on the homoeologous group 2 chromosomes of wheat. Rht_NM9has a negative effect on plant height and positive effects on effective tiller number and grain size, thus, Rht_NM9 could be used for elucidating the mechanisms underlying plant architecture and grain development.