Inhibition of plasma membrane redox activities and elongation growth of soybean

NADH-ferricyanide oxido-reductase (EC 1,6,99,3) of purified plasma membrane vesicles isolated by aqueous two-phase partition from segments of etiolated soybean [ Glycine max (L.) Merr. cv. Williams] hypocotyls was used as a measure of plasma membrane redox activity. Elongation growth of hypocotyl segments floated on the solutions was determined in parallel. Cis -platinum (II) diammine dichloride ( cis -platin), adriamycin and p -nitrophenylacetate, agents known to inhibit cell proliferation and plasma membrane redox activities in mammalian cells inhibited both NADH-ferricyanide oxido-reductase of the isolated membrane vesicles and elongation growth of intact hypocotyl segments. Auxin(2,4-dichlorophenoxyacetic acid)-induced growth of the isolated segments was inhibited preferentially at drug concentrations where control growth was affected only slightly. The findings suggest a connection between plasma membrane redox reactions and the control of elongation growth in plants.     

Use of dipyridyl-dithio substrates to measure directly the protein disulfide-thiol interchange activity of the auxin stimulated NADH: Protein disulfide reductase (NADH oxidase) of soybean plasma membranes

Dipyridyl-dithio substrates were cleaved by isolated vesicles of plasma membranes prepared from etiolated hypocotyls of soybean. The cleavage was stimulated by auxins at physiological concentrations. The substrates utilized were principally 2,2-dithiodippyrine (DTP) and 6,6-dithiodinicotinic acid (DTNA). The DTP generated 2 moles of 2-pyridinethione whereas the 6,6-dithiodinicotinic acid generated 2 moles of 6-nicotinylthionine. Both products absorbed at 340 nm. The auxin herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D) stimulated the activity approximately 2-fold to a maximum at about 10 M. Concentrations of 2,4-D greater than 100 M inhibited the activity. Indole-3-acetic acid stimulated the activity as well. The growth-inactive auxin, 2,3-dichlorophenoxyacetic acid (2,3-D), was without effect. DTNA cleavage correlated with oxidation of NADH and reduction of protein disulfide bonds reported earlier in terms of location at the external plasma membrane surface, absolute specific activity, pH dependence and auxin specificity. The dipyridyl-dithio substrates provide, for the first time, a direct measure of the disulfide-thiol interchange activity of the protein previously measured only indirectly as an auxin-dependent ability of isolated plasma membrane vesicles to restore activity to scrambled and inactive RNase.     

Chlorophyll-protein levels and degree of thylakoid stacking in radish chloroplasts from high-light, low-light and bentazon-treated plants

Lemna gibba plants were incubated aseptically on medium containing labelled 10^-7 M indole-3-acetic acid (IAA-1-¹⁴C). Most of the radioactivity disappeared from the culture medium during a 24 h light period. A high percentage of the loss was due to photolysis and only a low percentage of the radioactivity was recovered in the plants. Uptake of ¹⁴C by the plants was strongly stimulated by light. The radioactivity taken up by the plants was the sum of photosynthetically taken up ¹⁴CO₂ and ¹⁴C taken up in IAA. Analyses with the indolo-α-pyrone fluorescence method revealed that the free IAA content was almost the same in plants grown in control and in IAA media for 5 h, whereas the amount of IAA which could be liberated by alkaline hydrolysis was doubled by the presence of IAA in the medium.     

Indole-3-acetic acid oxidase and syringaldazine oxidase activities of peroxidase isozymes in soybean root nodules

Many isoperoxidases with indole-3-acetic acid oxidase (IAA) and syringaldazine oxidase activities were detected by polyacrylamide gel electrophoresis in soybean root nodules [ Glycine max (L.) Merrill, cv. Asgrow], detached at the onset of flowering. The kinetics of the two activities were studied with some of the isoperoxidases partially purified by ion exchange chromatography. IAA oxidase activity of the cationic isoforms showed a sigmoidal kinetic behaviour and a higher substrate affinity than the anionic ones, whereas typical saturation kinetics were found with an anionic fraction that contained leghemoglobins. So, nodule IAA oxidase activity may mainly be displayed by the cationic isoforms. These cationic isoperoxidases had high affinity towards syringaldazine and they also may be associated with cell wall rigidification.     

Metabolism of [14C]-indole-3-acetic acid by soybean callus and hypocotyl sections

Metabolism of indole-3-acetic acid in soybean [ Glycine max (L.) Merr.] was investigated with [1-¹⁴C]- and [2-¹⁴C]-indole-3-acetic acid (IAA) applied by injection into soybean hypocotyl sections and by incubation with soybean callus. Free IAA and its metabolites were extracted with 80% methanol and separated by high performance liquid chromatography with [³H]-IAA as an internal standard. Metabolism of IAA in soybean callus was much greater than that in tobacco ( Nicotiana tabacum L.) callus used for comparison. High performance liquid chromatography of soybean extracts showed at least 10 metabolite peaks including both decarboxylated and undecarboxylated products. A major unstable decarboxylated metabolite was purified. [¹⁴C]-indole-3-methanol (IM) was three times more efficient than [2-¹⁴C]-IAA as substrate for producing this metabolite. It was hydrolyzable by β-glucosidase (EC 3.2.1.21), yielding an indole and D-glucose. The indole possessed characteristics of authentic IM. Thus, the metabolite is tentatively identified as indole-3-methanol-β-D-glucopyranoside. The results suggest that soybean tissues are capable of oxidizing IAA via the decarboxylative pathway with indole-3-methanol-glucoside as a major product. The high rate of metabolism of IAA may be related to the observed growth of soybean callus with high concentrations of IAA in the culture medium.     

重金属镉（Cd）和增强UV—B辐射复合对大豆生长和生理代谢的影响

研究了大豆的生长、生物量、抗氧化酶活性和吲哚乙酸（IAA）氧化酶活性在Cd^2 、UV－B辐射和二者复合胁迫（Cd UV-B)下的变化。结果表明,Cd^2 和UV－B辐射都抑制大豆生长,并显著抑制根的伸长,二者复合后加强了对根伸长的抑制。UV－B辐射显著增强了POD、SOD活性,Cd^2 对POD活性影响不明显,但却拮抗UV－B对POD活性的诱导,SOD活性在各种胁迫下显著增强。虽然Cd%2 对叶片类黄酮含量影响不明显,但对UV－B诱导的类黄酮合成有一定影响。IAA氧化酶活性在复合作用下下降,可能是复合胁迫影响大豆生长的重要因素之一。     

Identification of a tumor inhibitory factor in rat ascites fluid

A polypeptide which inhibits the growth of human carcinoma cells has been characterized from Novikoff rat ascites fluid. This tumor inhibitory factor co-purified with transforming growth factor activity through acid/ethanol extraction and Bio-Gel chromatography. The two activities were completely separated by reverse phase HPLC. The tumor inhibitory factor is heat stable and requires disulfide bonds for bioactivity. This factor inhibited the anchorage independent growth of the more differentiated human colon carcinoma cell lines but did not affect the less differentiated carcinoma cells. The presence of stimulatory and inhibitory activities in the same extracts suggests that the relative concentrations of these factors may be important in the control of cell growth.     

镉(Cd)与UV-B复合胁迫影响大豆胚轴生长的生理调节机制研究

通过研究大豆胚轴生长及内源吲哚乙酸(IAA)、赤霉素(GAs)、过氧化物酶(POD)和吲哚乙酸氧化酶(IAA oxidase)活性变化对Cd、UV-B辐射和Cd UV-B(复合胁迫)的响应。分析了激素水平、酶活性变化以及胚轴生长变化特性。结果表明,UV—B辐射引起大豆上胚轴伸长减小;但Cd对上胚轴伸长无明显影响;Cd UV—B使上胚轴长度比UV—B作用时明显增加。UVB辐射显著降低了胚轴IAA含量;而GAs含量却显著升高;Cd胁迫下IAA和GAs变化并不明显;但Cd UV—B使IAA含量显著升高,而对GAs无明显影响。UVB辐射使IAA氧化酶和POD活性显著增强,而Cd对这两种酶活性影响并不明显;但Cd UV—B复合胁迫下胚轴的IAA水平较高。尽管UVB辐射引起胚轴中GAs含量显著增加,但研究结果显示IAA含量变化是胁迫下引起胚轴生长改变的更直接原因。研究还表明Cd UV—B时,大大削弱了UV—B辐射下IAA氧化酶活性增强,加之Cd对POD活性的抑制,导致复合胁迫下胚轴的IAA水平较高。证明复合胁迫可以改变单一胁迫下植物激素的调控机制。     

Effects of indole-3-acetic acid on arsenic uptake and antioxidative enzymes in Pteris cretica var. nervosa and Pteris ensiformis

A hydroponic experiment was conducted to investigate the effects of indole-3-acetic acid (IAA) on arsenic (As) uptake and antioxidative enzymes in fronds of Pteris cretica var. nervosa (As hyperaccumulator) and Pteris ensiformis (non-hyperaccumulator). Plants were exposed to 2 mg L^?1 As(III), As(V) or dimethylarsinic acid (DMA) and IAA concentrations for 14 d. The biomass and total As in the plants significantly increased at 30 mg L^?1 IAA. Superoxide dismutase (SOD) activities significantly increased with IAA addition. Catalase (CAT) activities showed a significant increase in P. ensiformis exposed to three As species at 30 or 50 mg L^?1 IAA but varied in P. cretica var. nervosa. Peroxidase (POD) activities were unchanged in P. ensiformis except for a significant decrease at 50 mg L^?1 IAA under As(III) treatment. However, a significant increase was observed in P. cretica var. nervosa at 10 mg L^?1 IAA under As(III) or DMA treatment and at 50 mg L^?1 IAA under As(V) treatment. Under DMA stress, malondialdehyde contents in fronds of P. cretica var. nervosa showed a significant decrease at 10 mg L^?1 IAA but remained unchanged in P. ensiformis. Therefore, IAA enhanced As uptake and frond POD activity in P. cretica var. nervosa under As stress.     

Relationships between xanthoxin,phototropism, and elongation growth in the sunflower seedling Helianthus annuus L.

For phototropic curvature of a green sunflower seedling, only the hypocotyl has to be illuminated; the tip and cotyledons are not involved in stimulus perception. The etiolated seedling is phototropically insensitive, illumination of only the hypocotyl renders it sensitive. It is concluded that the photoreceptor is located within the responding organ. In curving seedlings, the endogenous indoleacetic acid (IAA) remains evenly distributed. However, the inhibitor, xanthoxin (Xa), accumulates on the illuminated side. The degree of phototropic response is generally related to the concentration of Xa. The amount of phototropic curvature is independent of the rate of elongation growth, the former can be changed without affecting the latter, and vice versa. The data conflict with the Cholodny-Went theory, whereas they support the hypothesis of Blaauw that the phototropic reaction is caused by the local accumulation of a growth-inhibiting substance on the irradiated side.Abbreviations CCC chlormequat, (2-chloroethyl)trimethylammonium chloride - GA₃ gibberellic acid - IAA indole-3-acetic acid - Xa xanthoxin     

Promotion of stem elongation by indole-3-butyric acid in intact plants of Pisum sativum L.

While indole-3-butyric acid (IBA) has been confirmed to be an endogenous form of auxin in peas, and may occur in the shoot tip in a level higher than that of indole-3-acetic acid (IAA), the physiological significance of IBA in plants remains unclear. Recent evidence suggests that endogenous IAA may play an important role in controlling stem elongation in peas. To analyze the potential contribution of IBA to stem growth we determined the effectiveness of exogenous IBA in stimulating stem elongation in intact light-grown pea seedlings. Aqueous IBA, directly applied to the growing internodes via a cotton wick, was found to be nearly as effective as IAA in inducing stem elongation, even though the action of IBA appeared to be slower than that of IAA. Apically applied IBA was able to stimulate elongation of the subtending internodes, indicating that IBA is transported downwards in the stem tissue. The profiles of growth kinetics and distribution suggest that the basipetal transport of IBA in the intact plant stem is slower than that of IAA. Following withdrawal of an application, the residual effect of IBA in growth stimulation was markedly stronger than that of IAA, which may support the notion that IBA conjugates can be a better source of free auxin through hydrolysis than IAA conjugates. It is suggested that IBA may serve as a physiologically active form of auxin in contributing to stem elongation in intact plants.     

热胁迫对不同发育阶段大豆生长素和脱落酸含量的影响(英文)

大豆等植物体内细胞受热或受其它理化因素（如：重金属离子、乙醇、氨基酸类似物）、以及缺氧、DNA损伤、病毒感染等病理因素刺激后,促发应激反应,启动某些基因表达,能产生各种生理活性物质以及各种酶类,共同调控代谢过程和某些激素的活动,如：吲哚乙酸（IAA）、脱落酸（ABA）等。这些内源IAA和ABA共同作用,调节着大豆的抗逆性,从而影响着大豆的农艺性状。本试验对华北生态型的六个大豆栽培种,进行热激处理;取其第三片展开叶,测其内源IAA和ABA含量。这些品种分别是：早熟17,诱处4号,诱变31,耐阴黑豆、科丰6号和科丰34（Tan．1）。初花期,第一天热激（43～45℃,4h）后,它们的IAA和ABA水平均显著高于对照（30～33℃）（Fig．1）。然而,在连续一天热激后（43～45℃,4h／d）,大多数品种的IAA和ABA比第一天减少（Fig．2）。盛花期连续热激处理二天（43～45℃,4h／d）,IAA水平一般低于对照（3～33℃）,半数品种ABA水平也低于对照（Fig．3）。结荚期连续两天热激后（45℃,4h／d）,IAA和ABA含量均显著高于对照（30～33℃）（Fig．4）。     

Effect of cadmium on growth, proton extrusion and membrane potential in maize coleoptile segments

Cd accumulation, its effects on elongation growth of maize coleoptile segments, pH changes of their incubation medium and the membrane potential of parenchymal cells were studied. The Cd content increased significantly with exposure to increasing cadmium concentrations. Coleoptile segments accumulated the metal more efficiently in the range 10–100 μM Cd, than in the range 100–1000 μM Cd. Cd at concentrations higher than 1.0 μM produced a significant inhibition of both growth and proton extrusion. 100 μM Cd caused depolarization of the plasma membrane (PM) potential in parenchymal cells. The simultaneous treatment of maize coleoptile segments by indole-3-acetic acid (IAA) and Cd, counteracted the toxic effect of Cd on growth. Moreover, our data also showed that 100 μM Cd suppressed the characteristic IAA-induced hyperpolarization of the membrane potential, causing membrane depolarization. These results indicate that the toxic effect of Cd on growth of maize coleoptile segments might be, at least in part, caused via reduced PM H⁺-ATPase activity.     

Characterization of auxin-binding proteins from zucchini plasma membrane

We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948–4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N₃-7-³H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or mutimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may possess transporter or channel function.Abbreviations HPLC high-pressure liquid chromatography - IAA indole-3-acetic acid - azido-IAA 5-N₃-7-³H-IAA - pI isoelectric point - PM plasma membrane - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We thank R. Hopkins and I. Gelford for excellent technical work and our colleagues, especially T. Wolpert and D.L. Rayle, for many helpful discussions. This work was supported by grants to T.L.L. from National Science Foundation (DCB 8904114), National Aeronautics and Space Administration (NAGW 1253) and by a grant to D.L. Rayle and T.L.L. from U.S. Department of Agriculture (90-37261-5779). G.R.H. is supported by a National Aeronautics and Space Administration Predoctoral Fellowship (NGT 50455).     

Expression of a bacterial gene in transgenic tobacco plants confers resistance to the herbicide 2,4-dichlorophenoxyacetic acid

Plants resistant to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were produced through the genetic engineering of a novel detoxification pathway into the cells of a species normally sensitive to 2,4-D. We cloned the gene for 2,4-D monooxygenase, the first enzyme in the plasmid-encoded 2,4-D degradative pathway of the bacterium Alcaligenes eutrophus, into a cauliflower mosaic virus 35S promoter expression vector and introduced it into tobacco plants by Agrobacterium-mediated transformation. Transgenic tobacco plants expressing the highest levels of the monooxygenase enzyme exhibited increased tolerance to 2,4-D in leaf disc and seed germination assays, and young plants survived spraying with levels of herbicide up to eight times the usual field application rate. The introduction of the gene for 2,4-D monooxygenase into broad-leaved crop plants, such as cotton, should eventually allow 2,4-D to be used as an inexpensive post-emergence herbicide on economically important dicot crops.     

Indole-3-acetic acid: A widespread physiological code in interactions of fungi with other organisms

Plants as well as microorganisms, including bacteria and fungi, produce indole-3-acetic acid (IAA). IAA is the most common plant hormone of the auxin class and it regulates various aspects of plant growth and development. Thus, research is underway globally to exploit the potential for developing IAA-producing fungi for promoting plant growth and protection for sustainable agriculture. Phylogenetic evidence suggests that IAA biosynthesis evolved independently in bacteria, microalgae, fungi, and plants. Present studies show that IAA regulates the physiological response and gene expression in these microorganisms. The convergent evolution of IAA production leads to the hypothesis that natural selection might have favored IAA as a widespread physiological code in these microorganisms and their interactions. We summarize recent studies of IAA biosynthetic pathways and discuss the role of IAA in fungal ecology.     

Control of the activity of plant plasma membrane MgATPase by the viscosity of the aqueous phase

The activity of plant plasma membrane (PM) MgATPase (EC 3. 6. 1. 35) was studied in PM vesicles purified from spring wheat (Triticum aestivum L. cv. Drabant) roots, winter wheat (Triticum aestivum L. cv. Martonvásári-8) roots, and soybean (Glycine max L. cv. Williams) hypocotyls by aqueous polymer two-phase partitioning. MgATPase from spring wheat roots was assayed at 23°C (a) in the absence and presence of Triton X-100, (b) in the presence of either 1 mM or 3 mM MgATP^2?, and in the presence of increasing concentration of sucrose from 10 mM upto 1. 2 M. Activity of MgATPase in PM vesicles from winter wheat roots was measured at 21°C at sucrose concentrations of 15 mM–1. 1 M in the presence of 3 mM MgATP^2? and absence of Triton X-100 only. MgATPase activity from soybean hypocotyls was studied (a) in the absence and presence of Triton X-100, (b) both at 21 and 31°C, in the presence of 3 mM MgATP^2? and varying concentrations of sucrose between 10 mM and 1. 1 M. In all cases, independently of the assay conditions and the source of PM, the MgATPase activity decreased with increasing sucrose concentration. Latency of the MgATPase activity depended only slightly on the concentration of sucrose. The Q₁₀ value for the MgATPase activity from soybean hypocotyls (and thereby the Arrhenius activation energy of the enzymatic reaction) was independent of the sucrose concentration and of the presence of Triton X-100. At optimal assay conditions, the ATP-hydrolyzing activity of plant PM MgATPase (v) was inversely proportional to the m-th power of the viscosity of aqueous phase (η) as predicted by the modified Kramers'theory of enzymatic catalysis: v ∝ (1/η)^m, where m is an empirical parameter between 0 and 1. For the activity of MgATPase in the three species studied, m varied between 0. 5 and 1. 1 in good agreement with the theory. We thus conclude that (a) the activity of integral membrane enzyme-proteins may be controlled not only by the property(ies) of the membrane lipid phase but also by the viscosity of the aqueous phase in the vicinity of such enzymes, and (b) the determination of vesicle sidedness based on enzyme latency may need a minor revision. Our interpretation is in agreement with the molecular dynamics approach of enzymatic catalysis worked out for soluble enzymes.     

Up-regulation of plasma membrane-associated redox activities in neuronal cells lacking functional mitochondria

Mitochondria-deficient cells (rho(o) cells) survive through enhanced glycolytic metabolism in the presence of pyruvate and uridine. The plasma membrane redox system (PMRS) contains several NAD(P)H-related enzymes and plays a key role in maintaining the levels of NAD(+)/NADH and reduced coenzyme Q. In this study, rho(o) cells were used to investigate how the PMRS is regulated under conditions of mitochondrial dysfunction. rho(o) cells exhibited a lower oxygen consumption rate and higher levels of lactate than parental cells, and were more sensitive to glycolysis inhibitors (2-deoxyglucose and iodoacetamide) than control cells. However, they were more resistant to H(2)O(2), consistent with increased catalase activity and decreased oxidative damage (protein carbonyls and nitrotyrosine). PM-associated redox enzyme activities were enhanced in rho(o) cells compared to those in control cells. Our data suggest that all PMRS enzymes and biomarkers tested are closely related to the ability of the PMs to maintain redox homeostasis. These results illustrate that an up-regulated PM redox activity can protect cells from oxidative stress as a result of an improved antioxidant capacity, and suggest a mechanism by which neurons adapt to conditions of impaired mitochondrial function.     

Flooding resistance of Rumex species strongly depends on their response to ethylene: Rapid shoot elongation or foliar senescence

In excised Zea maus L. coleoptiles incubated in aerated media at high fresh weight per volume ratios, indole-3-acetic acid induces transient drops of extracellular pH. Based on the quantitative dependency of the response on the initial auxin concentration we developed a novel auxin bioassay, which allows reliable estimation of IAA concentrations between 10⁻⁸⁵ and 10⁻⁵M. Using the bioassay and complementary concentration measurements by IAA fluorescence we found the transient IAA-induced pH response paralleled by a decrease of auxin activity and concentration in the medium. This decline is rapid and starts immediately upon auxin addition, and insofar differs from the well known IAA degradation by epiphytic bacteria in long-term auxin tests. We conclude that the transient character of the auxin pH response is due to rapid IAA metabolism. The effect occurs under those experimental conditions that are necessary for reliable estimations of auxin-induced shifts of cell wall pH, which considerably complicates the interpretation of the results.