Comparison of the properties of plasmalemma vesicles purified from wheat roots by phase partitioning and by discontinuous sucrose gradient centrifugation

Plasmalemma vesicles were purified from 7-day-old wheat ( Triticum aestivum L. cv. Drabant) roots by a) discontinuous sucrose gradient (SG), b) aqueous polymer two-phase partitioning (PP) and c) SG followed by PP (SG + PP). SG-purified plasmalemma preparations were 2-3 fold more contaminated with mitochondria (cytochrome c oxidase, EC 1.9.3.1) when compared to PP and (SG + PP)-purified plasmalemma. The electrostatic surface properties, as measured by 9-aminoacridine fluorescence, were similar in PP and (SG + PP)-purified plasmalemma and different from SG-purified. The latency of the MG-ATPase measured with Triton X-100 was 51, 81 and 82% for SG-, PP- and (SG + PP)-purified plasmalemma vesicles, respectively. The higher latency of the ATPase (and lower specific activity in the absence of Triton X-100) in PP-purified and (SG + PP)-purified preparations was not due to an effect of PEG, since exposure of SG-purified preparations to PEG either in the wash medium or in the ATPase assay medium did not change ATPase activity. It is concluded that SG-purified plasmalemma vesicles are more contaminated than PP-purified preparations and that the former are likely to be leaky. They are, therefore, less suitable for use in studies of transport across the plasmalemma.     

Oxygen consumption by purified plasmalemma vesicles from wheat roots: Stimulation by NADH and salicylhydroxamic acid (SHAM)

Plasmalemma vesicles from wheat (Triticum aestivum L.) roots consumed O₂ and the addition of 1 mM NADH increased the rate ~ 3-fold (to 15-30 nmol O₂·mg⁻¹·min⁻¹). The NADH-dependent O₂ uptake was abolished by catalase. In the presence of salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase pathway in plant mitochondria, NADH-dependent O₂ consumption was stimulated 10–20-fold (to 200–400 nmol·mg^1̄·min⁻¹). Catalase also abolished this stimulation, which was KCN-sensitive but antimycin A-insensitive, and the production of H₂O₂ during SHAM-stimulated NADH-dependent O₂ uptake was demonstrated. Irrespective of the mechanism, SHAM-stimulated respiration by root plasmalemma makes it difficult to interpret results on root respiration obtained using KCN and SHAM.     

Causes and location of non-specific effects of SHAM on O2 uptake by wheat roots

Uptake of O₂ by whole, detached, root systems of wheat ( Triticum aestivum L. cv. Alexandria) was titrated with salicylhydroxamic acid (SHAM) in the presence and absence of cyanide. The resulting Q_all plot was non-linear indicating that SHAM was acting non-specifically. The nature of the non-specific effects was investigated in reverse titration experiments. Uptake of O₂ was titrated with KCN in the presence and absence of SHAM at 1 m M and 25 m M , which yielded Q_cy1 values of < 1 and > 1, respectively. The results suggest that at 25 m M , SHAM inhibits the cytochrome pathway, but at 1 m M it stimulates an O₂-consuming process which is likely to be a peroxidase. A SHAM-stimulated peroxidase could easily be washed from these roots. In vitro, the peroxidase was stimulated to a similar extent by low (1 m M ) and high (25 m M ) concentrations of SHAM. Failure to inhibit with high concentrations of SHAM distinguishes this peroxidase from those bitherto eluted from root tissue. Reverse titration experiments in the presence and absence of 1 m M SHAM indicated that there were no significant side effects of SHAM in root tips. These data are supported by the negligible peroxidase activity that was washed from this root fraction. In contrast, significant side effects occurred in vivo, and substantial peroxidase activity was measured in vitro, from sections 4–6 cm and 18–20 cm behind the seminal root apex. The greatest activity was found with the 4–6 cm section which may be associated with high rates of cell wall lignification. The implications of these results for measurements of root respiration are discussed.     

The effect of aluminium on respiration of wheat roots

The effects of aluminium ions on respiration of excised root apices from wheat (Triticum aestivum L. cv. Vulcan) and on isolated mitochondria have been investigated. Addition of 75μ M aluminium to the growth medium of 4-day-old seedlings inhibited O₂ uptake by excised root apices by 23 and 35% after 12 and 24 h, respectively. This decreased rate of respiration was initially caused by inhibition of the cytochrome pathway of mitochondrial electron transport. The cyanide-insensitive, alternative pathway was inhibited only after more prolonged exposure to aluminium. Mitochondria isolated from roots of aluminium-treated seedlings had reduced oxidative capacity with substrates that supply electrons to Complexes I and II, compared with mitochondria from roots of untreated control seedlings. The state 3 and state 4 rates of O₂ uptake and the uncoupled rates with these substrates were also inhibited when aluminium was added directly to reaction mixtures containing mitochondria isolated from untreated plants. In contrast, when aluminium was added to reaction mixtures oxidizing exogenous NADH, state 4 O₂ uptake was stimulated, whereas no effect was observed on the state 3 rate or the rate in the presence of uncoupler. The results suggest that aluminium initially affects electron flow through Complexes I and II, and that after more prolonged exposure, aluminium may also interact with other sites in mitochondria.     

Interaction between NADPH-cytochrome p-450 reductase and cytochrome p-450 in the membrane of phosphatidylcholine vesicles

Cytochrome P-450 and NADPH-cytochrome P-450 reductase, both purified from liver microsomes of phenobarbital-pretreated rabbits, have been incorporated into the membrane of phosphatidylcholine vesicles by the cholate dialysis method. The reduction of cytochrome P-450 by NADPH in this system is biphasic, consisting of two first-order reactions. The rate constant of the fast phase, in which 80–90% of the total cytochrome is reduced, increases as the molar ratio of the reductase to the cytochrome is increased at a fixed ratio of the cytochrome to phosphatidylcholine, suggesting that the rate-limiting step of the fast phase is the interaction between the reductase and the cytochrome. The rate constant of the fast phase also increases when the amount of phosphatidylcholine, relative to those of the two proteins, is decreased. This latter observation suggests that the interaction between the two proteins is effected by their random collision caused by their lateral mobilities on the plane of the membrane of phosphatidylcholine vesicles. The rate constant of the slow phase as well as the fraction of cytochrome P-450 reducible in the slow phase, on the other hand, remains essentially constant even upon alteration in the ratio of the reductase to the cytochrome or in that of the two proteins to phosphatidylcholine. No satisfactory explanation is as yet available for the cause of the slow-phase reduction of cytochrome P-450. The overall activity of benzphetamine N-demethylation catalyzed by the reconstituted vesicles responds to changes in the composition of the system in a similar way to the fast-phase reduction of cytochrome P-450, though the latter is not the rate-limiting step of the overall reaction.     

Effects of iron-EDTA on uroporphyrinogen oxidation by liver microsomes

Uroporphyrinogen oxidation by hepatic microsomes from chick embryos or mice pretreated with methylcholanthrene was increased by addition of iron-EDTA. This increase was partially prevented by catalase, mannitol, ketoconazole and piperonyl butoxide, whereas only ketoconazole and piperonyl butoxide inhibited the oxidation in the presence and absence of iron-EDTA. These data suggest that the oxidations of uroporphyrinogen in the presence and absence of added iron occur by different mechanisms.     

The absorption and translocation of diclofop-methyl and amitrole in wheat and oat roots

The absorption and translocation of diclofop-methyl (methyl 2-[4(2',4'-dichlorophenoxy)phenoxy]propanoate) was examined by using a specially designed treatment apparatus that separated excised roots or roots of seedlings into four zones. [¹⁴C]-Diclofop-methyl was absorbed along the entire root length of both wheat ( Triticum aestivum L.) and oat ( Avena sativa L.). In both species, absorption was greatest in the apical region of the root. Absorption by the apical region of wheat roots was more than three times greater than the basal portions, and more than twice as great as the apical region of oat roots. Less than 5% of the absorbed diclofop-methyl was translocated in both wheat and oat roots. Diclofop-methyl and diclofop(2-[4(2',4'-dichlorophenoxy)phenoxy]propanoic acid) were the predominant translocated forms. The absorption and translocation of amitrole (3-amino-1,2,4-triazole) were also examined. Amitrole was absorbed along the entire length of wheat roots and translocated primatily in the basipetal direction. The usefulness of the specially designed apparatus for biochemical and physiological studies is discussed.     

Comparison of K,MgATPases in purified plasmalemma from wheat and oat. – Substrate specificities and effects of pH, temperature and inhibitors

Plasmalemma from 8-day old oat ( Avena sativa L. cv. Brighton) and spring wheat ( Triticum aestivum L. cv. Drabant), grown in the dark at 18°C, was prepared from the 10000 g (10 min) – 30 000 g (60 min) root homogenate by two-phase separation in three steps with 6.5% (w/w) Dextran T 500 and 6.5% (w/w) polyethylene glycol 4 000. Biochemically and with respect to activation by Mg²⁺ as well as by (Mg²⁺+ K⁺), the oat preparations clearly appeared as ATPase(s) in the pH range 5–8. They showed high specificity for ATP, temperature optima between 38 and 40°C, and were inhibited by vanadate, DCCD (dicyclohexylcarbodiimide) and SH-reagents, but not by oligomycin, ammonium molybdate or ouabain. In contrast, the preparations from wheat contained more than one type of MgATPase/ nucleotidase, as revealed by complex dependence on both pH and temperature as well as by comparatively low specificity towards nucleotides. However, no unspecific phosphatase was present, and the effect of K⁺ over and above that of Mg²⁺ was almost as specific as in oat by all criteria used. The data available from this and earlier investigations from our group would indicate that the complex reactions of preparations of wheat plasmalemma may not be due to contamination but, rather, expressions of the many biological functions that must be associated with the plasmalemma in vivo and which may be located in sub-units that are more firmly attached to wheat than to oat plasmalemma.     

Plasma membrane lipid alterations induced by NaCl in winter wheat roots

A highly enriched plasma membrane traction was isolated by two phase partitioning from wheat roots ( Triticum aestivum L. cv. Vivant) grown with and without 100 m M NaCl. The lipids of the plasma membrane fraction were extracted and characterised. Phosphatidylcholine and phosphatidylethanolamine were the major phospholipids with lesser amounts of phosphandylinositol, phosphatidylglycerol, diphosphalidylglycerol, phosphatidic acid and phosphatidylseriae. NaCl decreased the total phospholipids and the phosphatidylcholine portion of the plasma membranes. Salt treatment had no effect on total sterols and glycolipids. but the relative abundance of the tree sterols was altered: cholesterol, stigma sterol and brassicasterol were significantly increased. Salt treatment resulted in an increase of the more planar/less planar ratio of the free sterols and in introduction of a double bond in the C₂₂ position in the side chain of stigma sterol and brassicasterol. The degree of fatty acid saturation of total phospholipids, phospha-tidylcholine and phosphatidylethanolamine was increased after salt treatment. These lipid changes are discussed in relation to the salt tolerance mechanism.     

Mechanisms of reaction of hematoxylin with aluminium-treated wheat roots

Hematoxylin stain is used for localization of aluminium in plant root tissue and is the basis of a rapid assay of relative At tolerance among wheat cultivars. In the present study, mechanisms by which hematoxylin might selectively stain Al-sensitive wheat roots have been examined. The results are consistent with the idea that in Al-sensitive cultivars, hematoxylin forms complexes with Al that precipitate with phosphate as AlPO₄ in intercellular spaces: (1) Al and P are co-localized in the cell wall region of the outer cortex of Al-stressed roots by using x-ray microanalysis: (2) the molybdenum blue histochemical stain for extracellular phosphate reveals areas of stain that parallel those observed with hematoxylin; (3) in vitro, the presence of phosphate in an Al-hematoxylin reaction mixture causes formation of precipitate when the P/Al ratio exceeds 1. 0. I suggest that selective hematoxylin staining of Al-sensitive wheat cultivars is the result of direct damage by Al to root cells, leading to leakage of phosphorus into the cell wall region. Cultivars whose roots are damaged by Al in this way are likely to be judged Al-sensitive when other criteria such as growth or crop yield are used.     

Lignin deposition induced by aluminum in wheat (Triticum aestivum) roots

We investigated the relation between the toxic effect of aluminum (Al) on root growth and the lignin deposition in wheat ( Triticum aestivum L. cvs Atlas 66 and Scout 66). In the Al-tolerant cultivar Atlas 66, control treatment without AlCl₃ at pH 4.75, cell length increased dramatically in the portion of the root that was 0.6 to 3.2 mm from the root cap junction (approximately 1.0 to 3.6 mm from the root tip). However, treatment with 20 μ M AlCl₃ for 24 and 48 h completely inhibited root elongation and markedly decreased the length and increased the diameter of the cells in the same portion of the root. Moreover, marked deposition of lignin was observed in the cells that corresponded to the portion 1.5 to 4.5 mm from the root tip in Atlas 66 roots treated with 20 μ M AlCl₃, while no deposition of lignin was detected in control roots. Treatment with 5 μ M AlCl₃ slightly inhibited root growth and there was no deposition of lignin in the root. On the other hand, in roots of the Al-sensitive cultivar Scout 66, treatment with 5 μ M AlCl₃ completely inhibited root growth and markedly induced deposition of lignin. These results suggest that lignification in the elongating region coincided with the extent of inhibition of root growth by Al in two wheat cultivars that differed in their sensitivity to Al.     

Several lanthanides activate malate efflux from roots of aluminium‐tolerant wheat

Many elements of the lanthanide series exist as trivalent cations in solution below pH 6. The present study was carried out to investigate whether lanthanides could stimulate malate efflux from wheat (Triticum aestivum L.) roots, as has been found for trivalent aluminium (Al) cations. Excised root apices treated with 100 µm of each of seven different lanthanide elements (lanthanum, praseodymium, europium, gadolinium, terbium, erbium, and ytterbium) stimulated malate efflux, with five‐ to fifty‐fold more malate being released from an Al‐tolerant wheat line than from a near‐isogenic Al‐sensitive line. As erbium stimulated the greatest malate efflux of the lanthanides tested, this response was characterized further. The characteristics of the erbium‐activated efflux were similar to the Al‐activated efflux described previously suggesting that both of these ions activate the same transport mechanism. The capacity for erbium‐activated malate efflux cosegregated with Al tolerance in wheat seedlings derived from a cross between Al‐sensitive and Al‐tolerant near‐isogenic lines. This is the first study to identify cations, other than Al, which can activate malate release from wheat roots. It also provides additional evidence that malate efflux from root apices is the primary mechanism for Al tolerance in wheat.     

Absorption spectra of highly purified liver microsomal cytochrome P-450 in non-equilibrium conformational states at low temperatures

Absorption spectra of highly purified liver microsomal cytochrome P-450 in non-equilibrium states were obtained at 77 K by reduction with trapped electrons, formed by gamma-irradiation of the water-glycerol matrix. In contrast to the equilibrium form of ferrous cytochrome P-450 with the heme iron in the high-spin state the non-equilibrium ferrous state has a low-spin heme iron. The absorption spectrum of the non-equilibrium ferrous cytochrome P-450 is characterized by two bands at 564 (-band) and 530 nm (-band). When the temperature is increased to about 278 K this non-equilibrium form of the reduced enzyme is relaxed to the corresponding equilibrium form with a single absorption band at 548 nm in the visible region characteristic for a high-spin heme iron.     

Effects of different levels of nitrogen fertilization on soil respiration during growing season in winter wheat (Triticum aestivum)

 在目前全球氮沉降不断增加的背景下, 研究农田土壤呼吸对氮沉降的响应有助于理解未来生态系统碳循环对全球变暖的潜在影响。为探讨不同施氮浓度对华东地区冬小麦(Triticum aestivum)生长期土壤呼吸的影响, 该实验设计了对照组(不施加氮肥)和3种浓度施氮处理组(低浓度施氮15 g·m^–2·a^–1, 中等浓度施氮30 g·m^–2·a^–1, 高浓度施氮45 g·m^–2·a^–1)。使用便携式土壤CO₂通量观测仪LI-8100测定不同施氮浓度处理下冬小麦生长期(2013年12月至2014年5月)的土壤呼吸速率, 并探讨土壤呼吸与土壤温度、湿度等环境因素的关系。结果表明: 低、中、高3种浓度施氮处理的土壤呼吸速率平均值分别为5.29、6.17和6.75 μmol·m^–2 ·s^–1, 与对照组(土壤呼吸速率平均值为4.90 μmol·m^–2·s^–1)相比, 分别增加了7.8%、23.6%和37.8%; 地上生物量分别增加39.9%、104.4%和200.2%, 并与冬小麦生长季的总土壤呼吸正相关。5 cm深度土壤的温度与土壤呼吸速率呈指数关系(p < 0.05), 土壤呼吸季节变化的65%–75%由土壤温度引起, 其温度敏感性为2.09–2.32。结果表明, 添加氮肥促进了植物的生长, 增加了生物量, 从而增加了冬小麦农田的土壤呼吸速率。     

微粒体细胞色素P-450、NADPH-细胞色素P-450还原酶的纯化与重组活性

经苯巴比妥钠诱导的雄性大白鼠的肝微粒体纯化的细胞色素P-450同功酶组份,经SDS-PAGE鉴定呈电泳纯,分子量为55kD。部分纯化的NADPH-细胞色素P-450还原酶,含72和77kD两个蛋白质组分。上述细胞色素P-450和NADPH-细胞色素P-450还原酶与卵磷脂制备的脂质体重组后的活性试验表明,对艾氏剂有环氧化作用,对环已烷有羟化作用,对溴氰菊酯的羟化作用微弱。当重组系统中缺少细胞色素P-450组份时,对环已烷不再起作用。同时还研究了纯化的细胞色素P-450的光谱特性。     

不同施氮水平对冬小麦生长期土壤呼吸的影响

在目前全球氮沉降不断增加的背景下, 研究农田土壤呼吸对氮沉降的响应有助于理解未来生态系统碳循环对全球变暖的潜在影响。为探讨不同施氮浓度对华东地区冬小麦(Triticum aestivum)生长期土壤呼吸的影响, 该实验设计了对照组(不施加氮肥)和3种浓度施氮处理组(低浓度施氮15 g·m^-2·a^-1, 中等浓度施氮30 g·m^-2·a^-1, 高浓度施氮45 g·m^-2·a^-1)。使用便携式土壤CO₂通量观测仪LI-8100测定不同施氮浓度处理下冬小麦生长期(2013年12月至2014年5月)的土壤呼吸速率, 并探讨土壤呼吸与土壤温度、湿度等环境因素的关系。结果表明: 低、中、高3种浓度施氮处理的土壤呼吸速率平均值分别为5.29、6.17和6.75 μmol·m^-2 ·s^-1, 与对照组(土壤呼吸速率平均值为4.90 μmol·m^-2·s^-1)相比, 分别增加了7.8%、23.6%和37.8%; 地上生物量分别增加39.9%、104.4%和200.2%, 并与冬小麦生长季的总土壤呼吸正相关。5 cm深度土壤的温度与土壤呼吸速率呈指数关系(p < 0.05), 土壤呼吸季节变化的65%-75%由土壤温度引起, 其温度敏感性为2.09-2.32。结果表明, 添加氮肥促进了植物的生长, 增加了生物量, 从而增加了冬小麦农田的土壤呼吸速率。     

A protein similar to PR (pathogenesis-related) proteins is elicited by metal toxicity in wheat roots

An acidic, low molecular weight protein called TA1-18 (T for Triticum. Al for aluminium and 18 for its approximate molecular weight) is induced in wheat roots that are exposed to growth-inhibiting concentrations of Al. Enhanced biosynthesis of TA1-18 began during the period 3 to 6 h after exposure to Al, and reached a maximum after 9 to 12 h of treatment. A protein with the same molecular weight and pl was also elicited during toxicity associated with Cu and Cd, with calcium deprivation, and low (3. 5) pH, but not by heat shock. TA1-18 was formed in small amounts in triticale, but was not detected in rye during exposure to growth-inhibiting levels of Al. Amino acid sequencing of trypsin fragments of TA1-18 revealed strong homology to pathogenesis-related protein PR2 from parsley cultures, with which TA1-18 also shares similar molecular weight and pl. Aluminium toxicity appears to have features in common with pathogenesis such that similar proteins are formed in response to both types of stress.     

小麦幼苗叶片抗氰呼吸对轻度水分胁迫的响应

小麦幼苗经－０．５ＭＰa聚乙二醇（ＰＥＧ－６０００）溶液暗中渗透迫０、６、１２、１８、２４、３０、３６、４２、４８h,叶片的总呼吸速率（Ｖt)呈现先上升后降低的趋势,交替途径呼吸也表现出相同的变化模式。水分胁迫初期（０－１２h),交替途径容量（Ｖalt)、实际运行活性（ρValt)及运行系数（ρ值）均上升,此后（１８－４８h）逐渐下降,水分胁迫也影响了呼吸电子流在２条呼吸途径中的分配比例,胁迫初期的０－１２h内,流经交替途径的电子流增多,而流向细胞色素主路的电子流减少,但随着胁迫时间的延长,交替途径的贡献降低,而细胞色素主路的贡献增加,说明小麦叶片的抗氰呼吸在水分胁迫初期被诱导增加,而随着胁迫进行的延长又表现为下降。     

Uptake of calcium in wheat and cucumber roots

Uptake of Ca²⁺(⁴⁵Ca) was investigated in plants of wheat ( Triticum aestivum L. var. Svenno) and cucumber ( Cucumis sativus L. var. Cilla) cultivated in a nutrient solution with various Ca²⁺ concentrations. The adsorption of Ca²⁺ was higher in cucumber roots than in wheat roots especially at lower Ca²⁺ levels in the external medium. The intracellular fraction of Ca²⁺ was less than 20% of the total Ca²⁺ in wheat roots and less than 10% of the total Ca²⁺ in cucumber roots. The uptake of Ca²⁺ in cucumber was about 40 times higher than in wheat. Transport of Ca²⁺ in the roots towards the endodermis is suggested to take place mainly in the apoplastic pathway regulated by the availability of negatively charged binding sites along the cell wall continuum. Further transport of Ca²⁺ towards the stele may involve diffusion of Ca²⁺ into the symplasm in the vicinity of the endodermis. An active extrusion of Ca²⁺ towards the stele or towards the external medium is suggested to play a role in the regulation of Ca²⁺ uptake.