从无芒稗花粉活力评价其与转基因抗除草剂水稻基因漂移的可能性

为给转基因抗除草剂水稻和稗草间的基因漂移研究提供必要的信息,探寻了无芒稗(Echinochloacrusgalli var.mitis)花粉活力及其测定的最佳方法,并利用该方法测定了无芒稗开花盛期后离体和活体条件下不同时间的花粉活力。结果表明无芒稗开花盛期取样离体条件下花粉活力下降较快,3 h后花粉活力只有5.41％,活体条件下花粉活力下降较慢,3 h后仍有17.31％的花粉有活力。这说明无芒稗花粉在水稻开花时仍有部分保持活力,因而存在潜在的基因漂移可能性。同时用最佳培养基法测定了不同条件处理下(紫外灯照射,反复冻融,黑暗放置)无芒稗花粉活力,结果表明无芒稗花粉在不利环境条件下活力难以保持。     

Evidence for plantlet regeneration via somatic embryogenesis in the grasses Echinochloa muricata and E. crus-galli var. Oryzicola

Explants for tissue culture were derived from mesocotyl plate tissue of Echinocha crus-galli var. oryzicola and E. muricata seedlings germinated under anaerobic conditions. Callus was initiated in the dark under aerobic conditions on a modified Murashige and Skoog medium plus 10 or 5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 2 mg/l of 6-benzyl-aminopurine (BAP). Transfer of this callus tissue into the light on MS medium containing low auxin (≤ 5 mg/l) readily resulted in the formation of green plantlets. Scanning electron microscopic observations revealed that regeneration occured through the formation of somatic embryros. Capacity for regeneration is maintained after repeated callus subculture. This regenerative capacity via somatic embyros provides a valuable research system for continuing the study of the metabolism and developmental physiology of Echinochloa.     

大型水库消落带中西来稗种子耐淹性沿高程梯度的种内分化

大型水库消落带中西来稗种子耐淹性沿高程梯度的种内分化 在水文节律稳定、每年蓄水时间很长且水位涨落落差很大的水库中,生长在水库库岸消落带不同高程区域中的植物每年要面对长时间的不同强度的水淹,这种每年发生持续的水淹胁迫可能会导致水库库岸消落带中生长的植物的种子耐淹能力发生种内分化。西来稗(Echinochloa crusgalli var. zelayensis)是在三峡水库消落带中自然生长的优势一年生植物,在三峡水库消落带植被及消落带生态环境保护中发挥着重要作用。本研究的目的在于探究三峡水库消落带中生长的西来稗种子的耐淹能力是否已发生种内分化,以及此分化的发生是否与较弱的种子扩散能力有关。在本研究中,采集来自三峡水库消落带不同高程区域中生长的西来稗种子,在三峡水库即将蓄水时将所有采集的种子放置于三峡水库消落带中4个不同高程位置接受不同深度不同时长的水库蓄水水淹,同时把所有采集的种子也放置于不会被水库蓄水淹没的非消落带河岸上作为对照处理;测定种子在水中的漂浮持续时间和蓄水期间水库水位上涨速度以量化在水库蓄水水位上涨时种子的扩散能力,水库蓄水结束水退后检测放置于水库消落带和非消落带河岸上的种子的种子完好率、种子萌发能力和成苗率。研究结果发现,在经历三峡水库蓄水水淹后,西来稗种子的完好率和最终成苗率随种子母株在消落带中所处的高程的增加而显著降低,表明在三峡水库成库运行七年后,在三峡水库消落带中生长的西来稗的种子耐淹能力已发生了种内分化。三峡水库蓄水水位上涨时西来稗种子在水中较短的漂浮持续时间和水库蓄水时较慢的水位上升速度限制了西来稗种子的水媒传播和扩散距离,从而有利于西来稗种子的耐淹能力发生种内分化。     

土壤中乙草胺的微生物降解及其对防除稗草持效性的影响

采用气相色谱法和生物测定法,研究了土壤中乙草胺的微生物降解及其对防除稗草持效性的影响.结果表明,在同样的湿度和温度条件下,当添加到土壤中的乙草胺浓度为125、25和5.0 mg·kg^-1时,相同浓度的乙草胺在非灭菌土壤中的半衰期显著短于灭菌土壤,说明土壤微生物对乙草胺有明显的降解作用.三大主要菌群分离培养物降解实验与上述结果一致.生物测定结果表明,乙草胺在非灭菌土壤中防除稗草的持效期显著短于灭菌土壤,微生物的存在缩短了乙草胺在土壤中的滞留时间,从而降低了乙草胺防除稗草的持效性.     

Purification, Properties and Phosphorylation of Anaerobically Induced Enolase in Echinochloa phyllopogon and E. crus-pavonis

Enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11 [EC] ) activityis differentially induced by anoxia in the flood-tolerant speciesE. phyllopogon (Stev.) Koss and the flood-intolerant speciesE. crus-pavonis (H.B.K.) Schult. To examine the regulation ofenolase at the protein level, we purified the enzyme from bothspecies to near homogeneity and compared their physico-chemicaland catalytic properties. Enolase purified from E. phyllopogonexhibits optimal activity at pH 7.0, a K_m of 80 µM for2-PGA, a Q₁₀ of 1.97 and an E_a of 12.3 kcal mol^-1. Similarly,enolase from E. crus-pavonis exhibits optimal activity at pH7.0, a K_m of 50 µM for 2-PGA, a Q₁₀ of 2.04 and an E_aof 12.9 kcal mol^-1. The enzyme from both species is thermostable(100% active after 15 min, 50°C) and is a homodimer of 52.5kDa subunits as resolved by SDS-PAGE and immunoblotting. E.phyllopogon enolase was phosphorylated in vitro using either[     

Distribution of Cytochrome c Peroxidase Activity in Wild-Type and Petite Cells of Bakers'' Yeast Grown Aerobically and Anaerobically

Studies of mitochondrial biogenesis in yeast have been hampered by a lack of suitable membrane markers in anaerobically grown cells subsequently grown in air. Cytochrome c peroxidase activity and subcellular location was studied to determine whether it would be a useful marker for an analysis of mitochondrial formation. Cytochemical tests revealed enzyme reaction product on all mitochondrial membranes in aerobically grown wild-type cells. Anaerobically grown wild-type and all petite cultures contained cytochrome c peroxidase cytochemical reaction deposits on abundant cytoplasmic membranes and on the few mitochondrial profiles which also were seen in the electron photomicrographs. Biochemical studies corroborated the cytochemistry because mitochondrial fractions were greatly enriched in cytochrome c peroxidase activity for aerobically grown wild-type cultures, but petite and anaerobically grown wild-type cultures showed higher enzyme activities in supernatant fractions than was present in the corresponding particulate fractions after differential centrifugation. Evidence from low-temperature microspectroscopy, spectrophotometric assays of mitochondrial enzyme activities, and electron microscopy showed mitochondrial formation during the time required for preparation and lysis of spheroplasts from anaerobically grown cultures. The data were interpreted as indicating that cytochrome c peroxidase was an oxygen-inducible enzyme, and that there was a developmental relationship between enzyme-reactive membranes of mitochondria and cytoplasm during the period of respiratory adaptation.     

Effects of NaCl and Proline on Polyphenol Oxidase Activity in Bean Seedlings

In this work, the effects of NaCl (0, 50, 100, and 150 mM), proline (0, 5 and 10 mM) and NaCl + proline in combinations on activity of polyphenol oxidase (PPO; E.C. 1.10.3.1) and soluble protein content have been investigated in the root, stem and leaf tissues of bean (Phaseolus vulgaris L.) seedlings grown in embryo culture. PPO activities were higher in all the tissues treated with NaCl, proline and NaCl + proline combinations those that of the control tissues. The protein content was very high in tissues exposed to proline and NaCl + proline combination, but NaCl alone decreased protein contents in root and leaf tissues. The results suggest that proline may play a role as an enzyme-stabilizing agent in salt stress.     

The Escherichia coli CydX Protein Is a Member of the CydAB Cytochrome bd Oxidase Complex and Is Required for Cytochrome bd Oxidase Activity

Cytochrome bd oxidase operons from more than 50 species of bacteria contain a short gene encoding a small protein that ranges from ∼30 to 50 amino acids and is predicted to localize to the cell membrane. Although cytochrome bd oxidases have been studied for more than 70 years, little is known about the role of this small protein, denoted CydX, in oxidase activity. Here we report that Escherichia coli mutants lacking CydX exhibit phenotypes associated with reduced oxidase activity. In addition, cell membrane extracts from ΔcydX mutant strains have reduced oxidase activity in vitro. Consistent with data showing that CydX is required for cytochrome bd oxidase activity, copurification experiments indicate that CydX interacts with the CydAB cytochrome bd oxidase complex. Together, these data support the hypothesis that CydX is a subunit of the CydAB cytochrome bd oxidase complex that is required for complex activity. The results of mutation analysis of CydX suggest that few individual amino acids in the small protein are essential for function, at least in the context of protein overexpression. In addition, the results of analysis of the paralogous small transmembrane protein AppX show that the two proteins could have some overlapping functionality in the cell and that both have the potential to interact with the CydAB complex.     

Isolation and Characterization of Rhodobacter capsulatus Mutants Affected in Cytochrome cbb3 Oxidase Activity

The facultative phototrophic bacterium Rhodobacter capsulatus contains only one form of cytochrome (cyt) c oxidase, which has recently been identified as a cbb₃-type cyt c oxidase. This is unlike other related species, such as Rhodobacter sphaeroides and Paracoccus denitrificans, which contain an additional mitochondrial-like aa₃-type cyt c oxidase. An extensive search for mutants affected in cyt c oxidase activity in R. capsulatus led to the isolation of at least five classes of mutants. Plasmids complementing them to a wild-type phenotype were obtained for all but one of these classes from a chromosomal DNA library. The first class of mutants contained mutations within the structural genes (ccoNOQP) of the cyt cbb₃ oxidase. Sequence analysis of these mutants and of the plasmids complementing them revealed that ccoNOQP in R. capsulatus is not flanked by the oxygen response regulator fnr, which is located upstream of these genes in other species. Genetic and biochemical characterizations of mutants belonging to this group indicated that the subunits CcoN, CcoO, and CcoP are required for the presence of an active cyt cbb₃ oxidase, and unlike in Bradyrhizobium japonicum, no active CcoN-CcoO subcomplex was found in R. capsulatus. In addition, mutagenesis experiments indicated that the highly conserved open reading frame 277 located adjacent to ccoNOQP is required neither for cyt cbb₃ oxidase activity or assembly nor for respiratory or photosynthetic energy transduction in R. capsulatus. The remaining cyt c oxidase-minus mutants mapped outside of ccoNOQP and formed four additional groups. In one of these groups, a fully assembled but inactive cyt cbb₃ oxidase was found, while another group had only extremely small amounts of it. The next group was characterized by a pleiotropic effect on all membrane-bound c-type cytochromes, and the remaining mutants not complemented by the plasmids complementing the first four groups formed at least one additional group affecting the biogenesis of the cyt cbb₃ oxidase of R. capsulatus.The gram-negative facultative photosynthetic bacterium Rhodobacter capsulatus has a highly branched electron transport chain, resulting in its ability to grow under a wide variety of conditions (52). Its light-driven photosynthetic electron transfer pathway is a cyclic process between the photochemical reaction center and the ubihydroquinone cytochrome (cyt) c oxidoreductase (cyt bc₁ complex) (30). On the other hand, the respiratory electron transfer pathways of R. capsulatus are branched after the quinone pool and contain two different terminal oxidases, previously called cyt b₄₁₀ (cyt c oxidase) and cyt b₂₆₀ (quinol oxidase) (3, 27, 29, 53). The branch involving cyt c oxidase is similar to the mitochondrial electron transfer chain in that it depends on the cyt bc₁ complex and a c-type cyt acting as an electron carrier. The quinol oxidase branch circumvents the cyt bc₁ complex and the cyt c oxidase by taking electrons directly from the quinone pool to reduce O₂ to H₂O. The pronounced metabolic versatility, including the ability to grow under dark, anaerobic conditions (50, 52), makes these purple non-sulfur bacteria excellent model organisms for studying microbial energy transduction.Marrs and Gest (29) have reported the first R. capsulatus mutants which were defective in the respiratory electron transport chain. Of these mutants, M5 was incapable of catalyzing the α-naphthol plus N′,N′-dimethyl-p-phenylenediamine (DMPD) plus O₂→indophenol blue plus H₂O reaction (NADI reaction) and unable to grow by respiration (Res⁻), and hence was deficient in both terminal oxidases. Another mutant, M4, was also NADI⁻ but Res⁺ due to the presence of an active quinol oxidase. Marrs and Gest have also described two different spontaneous revertants of M5, called M6 and M7, which regained the ability to grow by respiration (29). M6 regained cyt c oxidase activity and became concurrently NADI⁺ and sensitive to low concentrations of cyanide and the cyt bc₁ inhibitor myxothiazol, but remained quinol oxidase⁻. On the other hand, M7 regained the quinol oxidase activity but remained cyt c oxidase⁻ (thus, NADI⁻ and resistant to myxothiazol, a phenotype identical to that of M4). All of these mutants remained proficient for phototrophic (Ps) growth.The cyt c oxidase of R. capsulatus has been purified previously and characterized as being a novel cbb₃-type cyt c oxidase without a Cu_A center (15). It is composed of at least a membrane-integral b-type cyt (subunit I [CcoN]) with a low-spin heme b and a high-spin heme b₃-Cu_B binuclear center, and two membrane-anchored c-type cyts (CcoO and CcoP). It has a unique active site that possibly confers a very high affinity for its substrate oxygen (49). The structural genes of this enzyme (ccoNOQP) have been sequenced recently from R. capsulatus 37b4 (45) and aligned to the partial amino acid sequence of the purified enzyme from R. capsulatus MT1131 (15). Although a ccoN mutant of strain 37b4 was reported to lack cyt c oxidase activity (45), the observed discrepancies between the amino acid sequence and the nucleotide sequence do not entirely exclude the possible presence of two similar cb-type cyt c oxidases in this species. The presence of a similar cyt c oxidase has also been demonstrated in several other bacteria, including P. denitrificans (9), R. sphaeroides (13), and Rhizobium spp. In the latter species, the homologs of ccoNOQP have been named fixNOQP (23, 34) and are required to support respiration under oxygen-limited growth during symbiotic nitrogen fixation (36).The biogenesis of a multisubunit protein complex containing several prosthetic groups, such as cyt cbb₃ oxidase, is likely to require many accessory proteins involved in various posttranslational events, including protein translocation, assembly, cofactor insertion, and maturation (46). Thus, insights into this important biological process, about which currently little is known, may be gained by searching for mutants defective in cyt c oxidase activity. In this work, we describe the isolation of such mutants and their molecular genetic characterization, including those already available, such as M4, M5, and M7G. These studies indicate that in R. capsulatus, gene products of at least five different loci are involved in the formation of an active cyt cbb₃ oxidase.     

大豆初生幼苗多胺氧化酶活性的细胞化学定位

对大豆(Glycinemax(L.)Merril“l)垦农4号”萌发种子和初生幼苗中的多胺氧化酶(polyamineoxidase,PAO,EC1.4.3.6)的活性和分布进行了研究。结果表明,PAO活性仅在种子萌发起始后(吸胀后24h)才检测到,然后随着种子萌发进程,PAO活性快速升高。但是,在萌发种子(吸胀后72h)和初生幼苗(吸胀后120h)中,PAO活性在各器官中的分布有明显差异。在萌发种子中,PAO活性在胚根最高(5.17±0.91Ug-1FW),胚轴次之,胚芽再次之,子叶活性最低(0.12±0.03Ug-1FW);在初生幼苗中,PAO活性在下胚轴中最高(5.47±0.66Ug-1FW),幼根次之,顶芽再次之,子叶最低(0.10±0.03Ug-1FW)。这种差异对种子萌发和幼苗形态建成有积极意义。运用细胞化学定位在透射电镜下观察初生幼苗PAO在各部位的分布,发现PAO主要定位在顶芽细胞的液泡膜上、子叶细胞的细胞壁及其外侧表面、下胚轴细胞的细胞壁及其表面,且PAO与细胞壁表面结合较紧;根细胞的细胞壁、细胞间隙、细胞膜、液膜上均有分布,但以液泡膜分布居多。本研究结果进一步证实了PAO在细胞壁和细胞间隙有着较广泛的分布。首次报道PAO在细胞膜和液泡膜上有分布。     

NaCl-Induced Changes in Putrescine Content and Diamine Oxidase Activity in Roots of Rice Seedlings

The effects of NaCl on putrescine (Put) content and diamine oxidase (DAO) activity in roots of rice seedlings were examined. NaCl treatment lowered the content of Put and increased the activity of DAO in roots. Our current results indicate that Cl^– is not required for NaCl-induced decline in Put content and increase in DAO activity in roots. Put content in roots of rice seedlings exposed to NaCl is possibly regulated by DAO activity.     

Development of Photosynthetic Activity following Anaerobic Germination in Rice-Mimic Grass (Echinochloa crus-galli var oryzicola)

Shoots of anaerobically germinated Echinochloa crus-galli var oryzicola are nonpigmented whether germinated in light or dark, and chlorophyll synthesis is minimal for the first 12 to 18 hours of greening after exposure to ambient conditions. When chlorophyll development is compared between greening anoxic and etiolated shoots, there is a 100-fold difference in chlorophyll levels at 8 hours, an 8-fold difference at 24 hours, but roughly equal amounts at 60 hours. The chlorophyll a/b ratio approaches 3 earlier in greening anoxic shoots than in greening etiolated shoots, relative to total chlorophyll. The long lag in chlorophyll synthesis can be shortened by giving dark-grown anoxic shoots a 24-hour midtreatment of air before light.

Development of photosynthetic activity in etiolated shoots, determined by CO₂ gas exchange, ¹⁴CO₂ uptake, and activity of carboxylating enzymes closely parallels development of chlorophylls. However, development of photosynthetic capability in greening anoxic shoots does not parallel chlorophyll development; ability to fix carbon lags behind chlorophyll synthesis. A reason for this lag is the very low activity of RuBP carboxylase during the first 36 hours of greening in anoxic shoots. The activity of phosphoenolpyruvate carboxylase is also delayed, but its kinetics more closely match those of chlorophyll development.

     

Diabetic Modulation of the Temperature Kinetics Properties of Cytochrome Oxidase Activity in Rat Brain Mitochondria

The effects of alloxan-diabetes and subsequent treatment with insulin on temperature kinetics properties of cytochrome oxidase activity from rat brain mitochondria were examined. The enzyme activity decreased only at the late stage of diabetes which was not normalized by insulin treatment; however at early stage of diabetes hyper-stimulation occurred. In the control animals the Arrhenius plot was chair shaped with three energies of (E₁, E₂ and E₃) and two phase transition temperatures (T_t1 and T_t2). At early diabetic stage the Arrhenius plot became biphasic and E₁ and E₂ decreased_; insulin treatment reversed chair-shaped pattern with increase in E₂. These changes correlated with transient changes in the phospholipids profiles especially decreased acidic phospholipids. The temperature kinetics parameters were minimally affected at the late stage of diabetes or by insulin treatment. Thus at the late stage the brain tissue seems to have readjusted to its insulin homeostasis.     

Development of Ascorbate Oxidase Activity and Its Iso-enzyme Pattern in the Roots of Pea Seedlings

Early in the germination of peas, ascorbate Oxidase activity increased markedly in the root and epicotyl, but not in the cotyledons. The activity in the root was suppressed by the administration of 6-methylpurine, thiouracil, cyclohexi-mide, thioproline, and phenylthiourea. Although enzyme development was inhibited by phenylthiourea and thioproline, root elongation was not affected by these reagents. As germination progressed, the ascorbate oxidase activity in the extract of the seedling's root could be separated into at least 3 to 7 isoenzymes.     

Homocysteine Restricts Copper Availability Leading to Suppression of Cytochrome C Oxidase Activity in Phenylephrine-Treated Cardiomyocytes

Cardiomyocyte hypertrophy induced by phenylephrine (PE) is accompanied by suppression of cytochrome c oxidase (CCO) activity, and copper (Cu) supplementation restores CCO activity and reverses the hypertrophy. The present study was aimed to understand the mechanism of PE-induced decrease in CCO activity. Primary cultures of neonatal rat cardiomyocytes were treated with PE at a final concentration of l00 µM in cultures for 72 h to induce cell hypertrophy. The CCO activity was determined by enzymatic assay and changes in CCO subunit COX-IV as well as copper chaperones for CCO (COX17, SCO2, and COX11) were determined by Western blotting. PE treatment increased both intracellular and extracellular homocysteine concentrations and decreased intracellular Cu concentrations. Studies in vitro found that homocysteine and Cu form complexes. Inhibition of the intracellular homocysteine synthesis in the PE-treated cardiomyocytes prevented the increase in the extracellular homocysteine concentration, retained the intracellular Cu concentration, and preserved the CCO activity. PE treatment decreased protein concentrations of the COX-IV, and the Cu chaperones COX17, COX11, and SCO2. These PE effects were prevented by either inhibition of the intracellular homocysteine synthesis or Cu supplementation. Therefore, PE-induced elevation of homocysteine restricts Cu availability through its interaction with Cu and suppression of Cu chaperones, leading to the decrease in CCO enzyme activity.     

Amine Oxidase from Lentil Seedlings: Energetic Domains and Effect of Temperature on Activity

Copper/TPQ amine oxidases from mammalian and plant sources have shown many differences in substrate specificity and molecular properties. In this work the activity of lentil seedling amine oxidase was followed at various temperatures in 100 mM potassium phosphate buffer, pH 7, using benzylamine as substrate. The discontinuous Arrhenius plot of lentil amine oxidase showed two distinct phases with a jump between them. Thermal denaturation of the enzyme, using differential scanning calorimetry under the same experimental conditions, showed a transition at the same temperature ranges in the absence of substrate, indicating the occurrence of conformational changes, with an enthalpy change of about 175.9 kJ/mole. The temperature-induced changes of the activity of lentil amine oxidase are compared with those of bovine serum amine oxidase (taken from the literature).