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.     

Nitrogen and Photosynthesis in the Flag Leaf of Wheat (Triticum aestivum L.)

Wheat (Triticum aestivum L. cv Yecora 70) plants were grown with various concentrations of nitrate nitrogen available to the roots. Sampling of flag leaves began after they had reached full expansion and continued throughout senescence. Rates of gas exchange, ribulose-1,5-bisphosphate (RuP₂) carboxylase activity, and the amounts of chlorophyll, soluble protein, nitrogen, and phosphorus were determined for each flag leaf. Rate of CO₂ assimilation was uniquely related to total leaf nitrogen irrespective of nutrient treatment, season, and leaf age. Assimilation rate increased with leaf nitrogen, but the slope of the relationship declined markedly when leaf nitrogen exceeded 125 millimoles nitrogen per square meter. Chlorophyll content and RuP₂ carboxylase activity were approximately proportional to leaf nitrogen content. As leaves aged, RuP₂ carboxylase activity and calculated Hill activity declined in parallel. With normal ambient partial pressure of CO₂, the intercellular partial pressure of CO₂ was always such that rate of assimilation appeared colimited by RuP₂ carboxylation and RuP₂ regeneration capacity.

The initial slope of rate of CO₂ assimilation against intercellular partial pressure of CO₂ varied nonlinearly with carboxylase activity. It is suggested that this was due to a finite conductance to CO₂ diffusion in the wall and liquid phase which causes a drop in CO₂ partial pressure between the intercellular spaces and the site of carboxylation. A double reciprocal plot was used to obtain an estimate of the transfer conductance.

     

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.     

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.     

High Frequency Divisions in Leaf Base Protoplasts of Wheat (Triticum aestivum L.)

Protoplasts were isolated from the basal meristematic region of leaves from 6-day-old seedlings of wheat (Triticum aestivum). Protoplasts divided when cultured on MS medium (as agarose beads) in presence of nurse tissue. Donor seedlings when grown on BAP-supplemented MS medium were found to be considerably superior for protoplast isolation and culture than when grown on MS basal medium, in terms of protoplast viability, cell wall formation and cell division frequency. In addition, reduction of ammonium content of the culture medium, together with a dark Incubation, led to a high protoplast division frequency of about 70%. Microcolonies of 10-to 12-celled stages were obtained in Triticum aestivum, varieties HD 2329, HD 2285, Kalyan Sona, Arjun and CPAN 1676.     

Purification and Properties of Cystathionine [gamma]-Synthase from Wheat (Triticum aestivum L.)

Cysthathionine [gamma]-synthase (CS), an enzyme involved in methionine biosynthesis, was purified from an acetone powder prepared from wheat (Triticum aestivum L.). After several chromatographic steps and radiolabeling of the partially purified enzyme with sodium cyanoboro[3H]hydride, a single polypeptide with a molecular weight of 34,500 was isolated by sodium dodecyl sulfate-high performance electrophoresis chromatography. Since the molecular weight of the native enzyme was 155,000, CS apparently consists of four identical subunits. The pyridoxal 5[prime]-phosphate-dependent forward reaction has a pH optimum of 7.5 and follows a hybrid ping-pong mechanism with Km values of 3.6 mM and 0.5 mM for L-homoserine phosphate and L-cysteine, respectively. L-Cysteine methyl ester, thioglycolate methyl ester, and sodium sulfide were also utilized as thiol substrates. The latter observation suggests that CS and phosphohomoserine sulfhydrase might be a single enzyme. CS does not seem to be a regulatory enzyme but was irreversibly inhibited by DL-propargylglycine (Ki = 45 [mu]M, Kinact = 0.16 min-1). Furthermore, the homoserine phosphate analogs 4-(phosphonomethyl)-pyridine-2-carboxylic acid, Z-3-(2-phosphonoethen-1-yl)pyridine-2-carboxylic acid, and DL-E-2-amino-5-phosphono-3-pentenoic acid acted as reversible competitive inhibitors with Ki values of 45, 40, and 1.1 [mu]M, respectively.     

小麦胆色素原脱氨酶的纯化及部分性质研究

生物中四吡咯化合物合成的共同途径是由δ－氨基酮戊酸（δ－ａｍｉｎｏｌｅｖｕｌｉｎｉｃａｃｉｄ,ＡＬＡ）在δ－氨基酮戊酸脱水酶（δ－ａｍｉｎｏｌｅｖｕｌｉｎａｔｅｄｅｈｙｄｒａｔａｓｅ,ＡＬＡＤ）作用下合成胆色素原（ｐｏｒｐｈｏ－ｂｉｌｉｎｏｇｅｎ,Ｐ...     

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.     

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     

Chlorophyll Fluorescence Detected Passively by Difference Reflectance Spectra of Wheat （Triticum aestivum L.） Leaf

The chlorophyll fluorescence （CF） signature emitted from vegetation provides an abundance of information regarding photosynthetics activity and has been used as a powerful tool to obtain physiological information of plant leaves in a non-invasive manner. CF is difficult to quantify because the CF signal is obscured by reflected light. In the present study, the apparent reflectance spectra of wheat （Triticum aestivum L.） leaves were measured under illuminations with and without filtering by three specially designed long-wave pass edge filters; the cut-off wavelengths of the three filters were 653.8, 678.2, and 694. l nm at 50% of maximum transmittance. The CF spectra could be derived as the reflectance difference spectra of the leaves under illuminations with and without the long wave pass edge filters. The ratio of the reflectance difference at 685 and 740 nm （Dif685/Dif740） was linear correlated with the CF parameters （maximal photochemical efficiency Fv/Fm, and the yield of quantum efficiency） measured by the modulated fluorometer. In addition, the ratio reflected the water stress status of the wheat leaf, which was very high when water deficiency was serious. This method provides a new approach for detecting CF and the physiological state of crops.     

Changes to the Stoichiometry of Glycine Decarboxylase Subunits during Wheat (Triticum aestivum L.) and Pea (Pisum sativum L.) Leaf Development

Changes in the levels of the four subunits of the mitochondrial enzyme glycine decarboxylase (EC 2.1.2.10) have been investigated during development in the 8 day old primary leaf of wheat (Triticum aestivum L.). Proteins were extracted from wheat leaf sections between the basal meristem and 8.5 centimeters. The individual glycine decarboxylase subunits were detected by Western blotting, using subunit-specific polyclonal antibodies, and quantified by laser densitometry. P, T, and H subunits showed similar developmental patterns along the leaf. All were below the level of detection up to 1.5 centimeters from the meristem, but then increased over the leaf length examined. In contrast, the increase in the L protein (lipoamide dehydrogenase) was more gradual, and levels in the youngest regions of the leaf were maintained at approximately 14% of those at 8.5 centimeters. In a complementary study, levels of the four subunits in light-grown leaf tissues were compared to those in etiolated leaves from wheat and pea (Pisum sativum L.), using the activity of the mitochondrial marker enzyme fumarase as the basis for comparison. For both wheat and pea, levels of P, T, and H proteins in etiolated tissues were between 25 and 30% of those in lightgrown tissue. However, in etiolated tissues L protein was present at levels of 60 to 70% of that in light-grown tissues. The results indicate that discrete mechanisms may control the synthesis of L, as compared to P, T, and H proteins.     

Floret Initiation and Development in Spring Wheat (Triticum aestivum L.)

The number and developmental stages of florets were determinedin each spikelet of the spike in the main shoots of spring wheat.Samples were taken frequently from plants grown in a phytotronand in a nitrogen application field-test. Ten stages of development,from floret initiation until anthesis, were recognized and described. Inter-spikelet variation in the total number of initiated floretswas rather small. However, the number of florets at advancedstages of development, as well as the number of grains, washighest in the central spikelets in which florets initiatedfirst. Floret initiation did not proceed beyond spike emergence,whereafter the distal florets and the spikelet apex degenerated.Grain-set was restricted to florets which had developed at leastto the stage of visible anther lobes at spike emergence. Thenumber of these florets was increased significantly by nitrogenapplication. Wheat, Triticum aestivum L., spikelet, floret, grain set, nitrogen     

部分耐盐小麦品种(系)SSR位点遗传多样性研究

选择有多态性的32对SSR引物对80个小麦耐盐品种(系)进行遗传差异研究,共检测出155个等位变异,平均每个位点上有4.75个等位变异;供试80份耐盐小麦品种(系)来源广泛,遗传基础丰富,表现出较高的遗传多样性,遗传相似系数范围在0.26～0.81;聚类分析结果显示,冬性小麦品种(系)聚为一大类;春性小麦品种(系)也聚为一大类;一些系谱相同或相近的品种(系)遗传相似系数较大;A、B、D基因组中SSR位点平均等位变异差异不大,以B基因组较高.     

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.     

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     

A Study of Floret Development in Wheat (Triticum aestivum L.)

Plants of wheat (Triticum aestlvum L.) cv. Aotea were grownat high or low nitrogen levels and in a natural photoperiodor continuous light. Starting 17–21 days from the double-ridgestage, eight plants from each treatment were sampled every 3days until anthesis, and the two basal, the sixth, and the terminalspikelets were sectioned longitudinally. A developmental scorewas assigned to each floret and rates of development calculated.Continuous light hastened development but reduced the numberof spikelets per ear, while high nitrogen delayed developmentbut increased spikelet numbers. The number of florets initiatedin each spikelet varied within narrow limits, but grain settingdepended strongly on spikelet position and on treatment. Althoughflorets were initiated in acropetal succession, the rate ofdevelopment tended to increase up to floret 4 but then declinedmarkedly. As a result grain setting was confined to basal floretpositions, although the two basal spikelets developed so slowlythat they contributed relatively little to grain yield. Distalflorets degenerated almost simultaneously at or before ear emergence,but those in intermediate positions continued to develop untilafter fertilization in the lower florets. It is argued thatthe spikelet is an integrated system in which correlative mechanismsplay a part throughout the development of the florets.     

In Vitro Synthesis of Wheat (Triticum aestivum L.) Storage Proteins

Free and membrane-associated polysomes were isolated in approximately equal amounts from endosperm of wheat kernels harvested 20 days after anthesis. The presence of heparin in the homogenizing buffer minimized polysome degradation. Ribonucleic acid from the isolated polysomes, when translated in vitro in a wheat germ system, yielded products ranging in size from about 12,000 to about 80,000 daltons, including at least two polypeptides that co-migrated with seed extract proteins in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The nature of the translation products of free and membrane-associated RNA are distinctly different, with membrane-associated RNA yielding a higher proportion of polypeptides in the size range of 30,000 to 37,000 daltons. Analysis of membrane-associated 3′-terminal polyadenylyl-containing RNA in vitro translation products, by solubility in 70% ethanol and by immunoprecipitation, indicates that the 33,000- to 37,000-dalton polypeptides contain gliadins, and the analysis provides evidence that these proteins are synthesized in association with membranous cell organelles. Gliadin polypeptides synthesized in vitro are larger than authentic gliadins and probably are precursors which, in vivo, undergo modification to yield the smaller final products.     

Properties of Pyrophosphate:Fructose-6-Phosphate Phosphotransferase from Endosperm of Developing Wheat (Triticum aestivum L.) Grains

Pyrophosphate:fructose-6-phosphate phosphotransferase (PFP, EC 2.7.1.90) from endosperm of developing wheat (Triticum aestivum L.) grains was purified to apparent homogeneity with about 52% recovery using ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose and gel filtration through Sepharose-CL-6B. The purified enzyme, having a molecular weight of about 170,000, was a dimer with subunit molecular weights of 90,000 and 80,000, respectively. The enzyme exhibited maximum activity at pH 7.5 and was highly specific for pyrophosphate (PPi). None of the nucleoside mono-, di- or triphosphate could replace PPi as a source of energy and inorganic phosphate (Pi). Similarly, the enzyme was highly specific for fructose-6-phosphate. It had a requirement for Mg²⁺ and exhibited hyperbolic kinetics with all substrates including Mg²⁺. K_m values as determined by Lineweaver-Burk plots were 322, 31, 139, and 129 micromolar, respectively, for fructose-6-phosphate, PPi, fructose-1,6-bisphosphate and Pi. Kinetic constants were determined in the presence of fructose-2,6-bisphosphate, which stimulated activity about 20-fold and increased the affinity of the enzyme for its substrates. Initial velocity studies indicated kinetic mechanism to be sequential. At saturating concentrations of fructose-2,6-bisphosphate (1 micromolar), Pi strongly inhibited PFP; the inhibition being mixed with respect to both fructose-6-phosphate and PPi, with K_i values of 0.78 and 1.2 millimolar, respectively. The inhibition pattern further confirmed the mechanism to be sequential with random binding of the substrates. Probable role of PFP in endosperm of developing wheat grains (sink tissues) is discussed.     

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.     

Protoplast Culture and Plant Regeneration of Winter Wheat (Triticum aestivum L.)

Suspension cultures were established from embryogenic calli derived from cultured anthers of cv. Jinghua No.1 and mature embryos of cv. Youmangbai No. 7, respectively. After being isolated and cultured in WPMI, protoplasts began to form cell walls within 1 day post-isolation, followed by cell division observed between 2–3 days. A division frequency of 22.0% was estimated on the 7th day of culture, and 43.7% on the 14th day. During 10–15 days after the initiation of culture, a large number of cell aggregates emerged, with 0.5–0.8% of plating efficiency. Protoplast-derived calli grew up to lmm or more in diameter when cultured for 4 weeks, and eventually gave rise to green plants through embryogenesis and organogenesis after being transferred to differentiation media. Plant regeneration from protoplasts was already obtained from Jinghua No.l, and protoplast-derived calli from Youmangbai No.7; an experiment on organ differentiation for the latter is under way. A few factors affecting the protoplast cultures were also studied.