Soybean Plasma Membrane Research Articles

The soybean plasma membrane GmDR1 protein conferring broad-spectrum disease and pest resistance regulates several receptor kinases and NLR proteins

Overexpression of Glycine max disease resistant 1 (GmDR1) exhibits broad-spectrum resistance against Fusarium virguliforme, Heterodera glycines (soybean cyst nematode), Tetranychus urticae (Koch) (spider mites), and Aphis glycines Matsumura (soybean aphids) in soybean. To understand the mechanisms of broad-spectrum immunity mediated by GmDR1, the transcriptomes of a strong and a weak GmDR1-overexpressor following treatment with chitin, a pathogen- and pest-associated molecular pattern (PAMP) common to these organisms, were investigated. The strong and weak GmDR1-overexpressors exhibited altered expression of 6098 and 992 genes, respectively, as compared to the nontransgenic control following chitin treatment. However, only 192 chitin- and 115 buffer-responsive genes exhibited over two-fold changes in expression levels in both strong and weak GmDR1-overexpressors as compared to the control. MapMan analysis of the 192 chitin-responsive genes revealed 64 biotic stress-related genes, of which 53 were induced and 11 repressed as compared to the control. The 53 chitin-induced genes include nine genes that encode receptor kinases, 13 encode nucleotide-binding leucine-rich repeat (NLR) receptor proteins, seven encode WRKY transcription factors, four ethylene response factors, and three MYB-like transcription factors. Investigation of a subset of these genes revealed three receptor protein kinases, seven NLR proteins, and one WRKY transcription factor genes that are induced following F. virguliforme and H. glycines infection. The integral plasma membrane GmDR1 protein most likely recognizes PAMPs including chitin and activates transcription of genes encoding receptor kinases, NLR proteins and defense-related genes. GmDR1 could be a pattern recognition receptor that regulates the expression of several NLRs for expression of PAMP-triggered immunity and/or priming the effector triggered immunity.

Roles of Soybean Plasma Membrane Intrinsic Protein GmPIP2;9 in Drought Tolerance and Seed Development.

Aquaporins play an essential role in water uptake and transport in vascular plants. The soybean genome contains a total of 22 plasma membrane intrinsic protein (PIP) genes. To identify candidate PIPs important for soybean yield and stress tolerance, we studied the transcript levels of all 22 soybean PIPs. We found that a GmPIP2 subfamily member, GmPIP2;9, was predominately expressed in roots and developing seeds. Here, we show that GmPIP2;9 localized to the plasma membrane and had high water channel activity when expressed in Xenopus oocytes. Using transgenic soybean plants expressing a native GmPIP2;9 promoter driving a GUS-reporter gene, it was found high GUS expression in the roots, in particular, in the endoderm, pericycle, and vascular tissues of the roots of transgenic plants. In addition, GmPIP2;9 was also highly expressed in developing pods. GmPIP2;9 expression significantly increased in short term of polyethylene glycol (PEG)-mediated drought stress treatment. GmPIP2;9 overexpression increased tolerance to drought stress in both solution cultures and soil plots. Drought stress in combination with GmPIP2;9 overexpression increased net CO2 assimilation of photosynthesis, stomata conductance, and transpiration rate, suggesting that GmPIP2;9-overexpressing transgenic plants were less stressed than wild-type (WT) plants. Furthermore, field experiments showed that GmPIP2;9-overexpressing plants had significantly more pod numbers and larger seed sizes than WT plants. In summary, the study demonstrated that GmPIP2;9 has water transport activity. Its relative high expression levels in roots and developing pods are in agreement with the phenotypes of GmPIP2;9-overexpressing plants in drought stress tolerance and seed development.

Simulations of simple linoleic acid-containing lipid membranes and models for the soybean plasma membranes.

The all-atom CHARMM36 lipid force field (C36FF) has been tested with saturated, monounsaturated, and polyunsaturated lipids; however, it has not been validated against the 18:2 linoleoyl lipids with an unsaturated sn-1 chain. The linoleoyl lipids are common in plants and the main component of the soybean membrane. The lipid composition of soybean plasma membranes has been thoroughly characterized with experimental studies. However, there is comparatively less work done with computational modeling. Our molecular dynamics (MD) simulation results show that the pure linoleoyl lipids, 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (18:0/18:2) and 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (di-18:2), agree very well with the experiments, which demonstrates the accuracy of the C36FF for the computational study of soybean membranes. Based on the experimental composition, the soybean hypocotyl and root plasma membrane models are developed with each containing seven or eight types of linoleoyl phospholipids and two types of sterols (sitosterol and stigmasterol). MD simulations are performed to characterize soybean membranes, and the hydrogen bonds and clustering results demonstrate that the lipids prefer to interact with the lipids of the same/similar tail unsaturation. All the results suggest that these two soybean membrane models can be used as a basis for further research in soybean and higher plant membranes involving membrane-associated proteins.

Simulation of Linoleoyl-Containing Pure Lipid Bilayer and Soybean Plasma Membranes

Molecular dynamics (MD) simulations have been extensively used to study lipid membranes in addition to experimental studies as they help better understand membranes in the atomic level. Computational models of bacterial (E. coli) membranes have been developed and applied to study the antimicrobial peptide proteins. Plant membranes are less frequently studied compared to the bacterial membrane. In this work, we will present the soybean plasma membranes models. The compositions of cell plasma membranes of soybean vary depending on the species, stage of development, and the part of the plant. The two parts of the plant that we study are the hypocotyl and the root. Each model consists of 100 lipids per leaflet, with the composition based on the weighted and averaged values from past experimental studies. Specifically, the hypocotyl membrane contains 7 types of unsaturated phospholipids and two types of sterols, while the root membrane contains 8 types of phospholipids and two types of sterols. All types of phospholipids in soybean contains the 18:2 (cis Δ9, 12) linoleoyl tail which was not well studied before, therefore, the simulations on the pure 18:0/18:2 and 18:2/18:2 phosphocholine (PC) lipid bilayers are also performed. The structural properties such as surface area per lipid, bilayer thicknesses, order parameters, and spin-lattice relaxation time are analyzed for all membranes. Moreover, the analyses of the sterols tilt angle distributions, hydrogen bonding, and clustering are also conducted for the soybean membranes. The structural properties of pure bilayers agree well with NMR experimental data validate the accuracy of 18:2 linoleoyl-containing lipids, based on which the soybean membrane models also result in reasonable structural properties. These results imply that the two soybean membrane models are realistic, and can facilitate the further study of soybean and other plant membranes.

Constitutive overexpression of soybean plasma membrane intrinsic protein GmPIP1;6 confers salt tolerance.

BackgroundUnder saline conditions, plant growth is depressed via osmotic stress and salt can accumulate in leaves leading to further depression of growth due to reduced photosynthesis and gas exchange. Aquaporins are proposed to have a major role in growth of plants via their impact on root water uptake and leaf gas exchange. In this study, soybean plasma membrane intrinsic protein 1;6 (GmPIP1;6) was constitutively overexpressed to evaluate the function of GmPIP1;6 in growth regulation and salt tolerance in soybean.ResultsGmPIP1;6 is highly expressed in roots as well as reproductive tissues and the protein targeted to the plasma membrane in onion epidermis. Treatment with 100 mM NaCl resulted in reduced expression initially, then after 3 days the expression was increased in root and leaves. The effects of constitutive overexpression of GmPIP1;6 in soybean was examined under normal and salt stress conditions. Overexpression in 2 independent lines resulted in enhanced leaf gas exchange, but not growth under normal conditions compared to wild type (WT). With 100 mM NaCl, net assimilation was much higher in the GmPIP1;6-Oe and growth was enhanced relative to WT. GmPIP1;6-Oe plants did not have higher root hydraulic conductance (Lo) under normal conditions, but were able to maintain Lo under saline conditions compared to WT which decreased Lo. GmPIP1;6-Oe lines grown in the field had increased yield resulting mainly from increased seed size.ConclusionsThe general impact of overexpression of GmPIP1;6 suggests that it may be a multifunctional aquaporin involved in root water transport, photosynthesis and seed loading. GmPIP1;6 is a valuable gene for genetic engineering to improve soybean yield and salt tolerance.

Oxygen activation at the plasma membrane: relation between superoxide and hydroxyl radical production by isolated membranes

Production of reactive oxygen species (hydroxyl radicals, superoxide radicals and hydrogen peroxide) was studied using EPR spin-trapping techniques and specific dyes in isolated plasma membranes from the growing and the non-growing zones of hypocotyls and roots of etiolated soybean seedlings as well as coleoptiles and roots of etiolated maize seedlings. NAD(P)H mediated the production of superoxide in all plasma membrane samples. Hydroxyl radicals were only produced by the membranes of the hypocotyl growing zone when a Fenton catalyst (FeEDTA) was present. By contrast, in membranes from other parts of the seedlings a low rate of spontaneous hydroxyl radical formation was observed due to the presence of small amounts of tightly bound peroxidase. It is concluded that apoplastic hydroxyl radical generation depends fully, or for the most part, on peroxidase localized in the cell wall. In soybean plasma membranes from the growing zone of the hypocotyl pharmacological tests showed that the superoxide production could potentially be attributed to the action of at least two enzymes, an NADPH oxidase and, in the presence of menadione, a quinone reductase.

Comparative analysis of soybean plasma membrane proteins under osmotic stress using gel‐based and LC MS/MS‐based proteomics approaches

To study the soybean plasma membrane proteome under osmotic stress, two methods were used: a gel-based and a LC MS/MS-based proteomics method. Two-day-old seedlings were subjected to 10% PEG for 2 days. Plasma membranes were purified from seedlings using a two-phase partitioning method and their purity was verified by measuring ATPase activity. Using the gel-based proteomics, four and eight protein spots were identified as up- and downregulated, respectively, whereas in the nanoLC MS/MS approach, 11 and 75 proteins were identified as up- and downregulated, respectively, under PEG treatment. Out of osmotic stress responsive proteins, most of the transporter proteins and all proteins with high number of transmembrane helices as well as low-abundance proteins could be identified by the LC MS/MS-based method. Three homologues of plasma membrane H(+)-ATPase, which are transporter proteins involved in ion efflux, were upregulated under osmotic stress. Gene expression of this protein was increased after 12 h of stress exposure. Among the identified proteins, seven proteins were mutual in two proteomics techniques, in which calnexin was the highly upregulated protein. Accumulation of calnexin in plasma membrane was confirmed by immunoblot analysis. These results suggest that under hyperosmotic conditions, calnexin accumulates in the plasma membrane and ion efflux accelerates by upregulation of plasma membrane H(+)-ATPase protein.

Origin of cadmium‐induced reactive oxygen species production: mitochondrial electron transfer versus plasma membrane NADPH oxidase

* Cadmium (Cd(2+)) is an environmental pollutant that causes increased reactive oxygen species (ROS) production. To determine the site of ROS production, the effect of Cd(2+) on ROS production was studied in isolated soybean (Glycine max) plasma membranes, potato (Solanum tuberosum) tuber mitochondria and roots of intact seedlings of soybean or cucumber (Cucumis sativus). * The effects of Cd(2+) on the kinetics of superoxide (O2*-), hydrogen peroxide (H(2)O(2)) and hydroxyl radical ((*OH) generation were followed using absorption, fluorescence and spin-trapping electron paramagnetic resonance spectroscopy. * In isolated plasma membranes, Cd(2+) inhibited O2*- production. This inhibition was reversed by calcium (Ca(2+)) and magnesium (Mg(2+)). In isolated mitochondria, Cd(2+) increased and H(2)O(2) production. In intact roots, Cd(2+) stimulated H(2)O(2) production whereas it inhibited O2*- and (*)OH production in a Ca(2+)-reversible manner. * Cd(2+) can be used to distinguish between ROS originating from mitochondria and from the plasma membrane. This is achieved by measuring different ROS individually. The immediate (<or= 1 h) consequence of exposure to Cd(2+) in vivo is stimulation of ROS production in the mitochondrial electron transfer chain and inhibition of NADPH oxidase activity in the plasma membrane.

Acoustic Technology for High-Performance Disruption and Extraction of Plant Proteins

Acoustic technology shows the capability of protein pellet homogenization from different tissue samples of soybean and rice in a manner comparable to the ordinary mortar/pestle method and far better than the vortex/ultrasonic method with respect to the resolution of the protein pattern through two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). With acoustic technology, noncontact tissue disruption and protein pellet homogenization can be carried out in a computer-controlled manner, which ultimately increases the efficiency of the process for a large number of samples. A lysis buffer termed the T-buffer containing TBP, thiourea, and CHAPS yields an excellent result for the 2D-PAGE separation of soybean plasma membrane proteins followed by the 2D-PAGE separation of crude protein of soybean and rice tissues. For this technology, the T-buffer is preferred because protein quantification is possible by eliminating the interfering compound 2-mercaptoethanol and because of the high reproducibility of 2D-PAGE separation.

A plasma membrane‐associated AAA‐ATPase from Glycine max

An AAA-ATPase (ATPases Associated with a Variety of Cellular Activities) localized to the plasma membrane of soybean (Glycine max) was isolated, partially sequenced and cloned (SBPM AAA-ATPase). The protein with an apparent monomer molecular mass of about 97 kDa was isolated using a combination of anion exchange, preparative SDS-PAGE, reverse phase HPLC, and ATP affinity chromatography. The cDNA for the full-length SBPM AAA-ATPase was cloned by screening an expression library using an antibody against the highly conserved Walker B AAA-ATP-binding motif. Northern blot analysis detected one transcript of approximately 2700 bp. The full-length cDNA sequence was that previously obtained (GenBank Database; U20213) encoding a protein with two copies of the conserved AAA-ATP-binding motif and regions of sequence homology with other AAA-ATPases. Electron microscopic preparations of the recombinant SBPM AAA-ATPase revealed hexamers typically formed by these proteins. The cloned and expressed protein was identical to the protein isolated from the soybean plasma membrane as confirmed using antisera raised to a non-conserved region of the derived protein sequence and by N-terminal sequencing of peptides derived from the isolated protein.

Auxin-activated NADH oxidase activity of soybean plasma membranes is distinct from the constitutive plasma membrane NADH oxidase and exhibits prion-like properties.

The hormone-stimulated and growth-related cell surface hydroquinone (NADH) oxidase activity of etiolated hypocotyls of soybeans oscillates with a period of about 24 min or 60 times per 24-h day. Plasma membranes of soybean hypocotyls contain two such NADH oxidase activities that have been resolved by purification on concanavalin A columns. One in the apparent molecular weight range of 14-17 kDa is stimulated by the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The other is larger and unaffected by 2,4-D. The 2,4-D-stimulated activity absolutely requires 2,4-D for activity and exhibits a period length of about 24 min. Also exhibiting 24-min oscillations is the rate of cell enlargement induced by the addition of 2,4-D or the natural auxin indole-3-acetic acid (IAA). Immediately following 2,4-D or IAA addition, a very complex pattern of oscillations is frequently observed. However, after several hours a dominant 24-min period emerges at the expense of the constitutive activity. A recruitment process analogous to that exhibited by prions is postulated to explain this behavior.

Immunochemical evidence for the presence of a lipoxygenase 1 in isolated plasma membranes from soybean cotyledons

An antibody was raised against a synthetic oligopeptide, corresponding to the 19–31 interval of the polypeptide chain of soluble lipoxygenase 1 (LOX 1) from soybean seeds. Cross-reactivity of this antibody towards proteins of a soybean cotyledon plasma membrane (PM) fraction was detected. The anti-LOX 1 antibody cross-reacted with a protein of approximately 94 kDa, when the membrane proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The same protein, separated in non-denaturing conditions, exhibited LOX activity with an optimum at pH 9.0. The cross-reactivity of the 94 kDa protein was maintained also in Na 2CO 3 or NaCl-washed membranes, confirming that the enzyme appears to be tightly bound. Finally, the protein, showing LOX activity, was separated by immunoprecipitation using the anti-LOX 1 antibody bound to protein A-Sepharose CL-4B. Again, reactivity with the protein of 94 kDa was detected. These findings show that isolated soybean PMs exhibit a LOX activity, which depends on the presence of the LOX 1 isoform. It is suggested that soluble LOX 1 may be in a dynamic equilibrium with a part of the enzyme, which appears to be linked to PMs.

Binding site for chitin oligosaccharides in the soybean plasma membrane.

Affinity cross-linking of the plasma membrane fraction to an (125)I-labeled chitin oligosaccharide led to the identification and characterization of an 85-kD, chitin binding protein in plasma membrane-enriched fractions from both suspension-cultured soybean cells and root tissue. Inhibition analysis indicated a binding preference for larger (i.e. degrees of polymerization = 8) N-acetylated chitin molecules with a 50% inhibition of initial activity value of approximately 50 nM. N-Acetyl-glucosamine and chitobiose showed no inhibitory effects at concentrations as high as 250 microM. It is noteworthy that the major lipo-chitin oligosaccharide Nod signal produced by Bradyrhizobium japonicum was also shown to be a competitive inhibitor of ligand binding. However, the binding site appeared to recognize the chitin portion of the Nod signal, and it is unlikely that this binding activity represents a specific Nod signal receptor. Chitooligosaccharide specificity for induction of medium alkalinization and the generation of reactive oxygen in suspension-cultured cells paralleled the binding activity. Taken together, the presence of the chitin binding protein in the plasma membrane fraction and the specificity and induction of a biological response upon ligand binding suggest a role for the protein as an initial response mechanism for chitin perception in soybean (Glycine max).

The plasma membrane NADH oxidase of soybean has vitamin K 1 hydroquinone oxidase activity

Isolated plasma membrane vesicles and the plasma membrane NADH oxidase partially purified from soybean plasma membrane vesicles exhibited a cyanide-insensitive vitamin K 1 hydroquinone oxidase activity with isolated plasma membrane vesicles. Reduced vitamin K 1 (phylloquinol) was oxidized at a rate of about 10 nmol/min/mg protein as determined by reduced vitamin K 1 reduction or oxygen consumption. The K m for reduced K 1 was 350 μM. With the partially purified enzyme, reduced vitamin K 1 was oxidized at a rate of about 600 nmol/min/mg protein and the K m was 400 μM. When assayed in the presence of 1 mM KCN, activities of both plasma membrane vesicles and of the purified protein were stimulated (0.1 μM) or inhibited (0.1 mM) by the synthetic auxin growth factor 2,4-dichlorophenoxyacetic acid. The findings suggest the potential participation of the plasma membrane NADH oxidase as a terminal oxidase of plasma membrane electron transport from cytosolic NAD(P)H via reduced vitamin K 1 to acceptors (molecular oxygen or protein disulfides) at the cell surface.

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'-dithiodipyridine (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 microM. Concentrations of 2,4-D greater than 100 microM 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.

NADH oxidase activity of soybean plasma membranes oscillates with a temperature compensated period of 24 min

Summary The enzymatic activity of a hormone‐stimulated and growth‐related protein disulfide‐thiol oxidoreductase (NADH oxidase) with protein disulfide‐thiol interchange activity of etiolated hypocotyls of soybean oscillates with a period of about 24 min or 60 times per 24 h day. The oscillations were temperature compensated such that the period remained constant at about 24 min between 17 and 37°C, a temperature range over which enzymatic activity varied approximately fourfold (Q10 of 2). The oscillations were observed with intact tissue sections, with isolated plasma membrane vesicles, and with the detergent‐solubilized and partially purified enzyme. The oscillations were observed both with the oxidation of NADH and in the restoration of activity to scrambled ribonuclease used as a measure of the protein disulfide‐thiol interchange activity. The enzymatic activity which is located at the cell surface may represent a biochemical reaction with a potential function as an ultradian oscillator of circadian time keeping.

Cloning of putative subunits of the soybean plasma membrane NADPH oxidase involved in the oxidative burst by antibody expression screening

Plant cells respond to a variety of external signals with the production of reactive-oxygen species. The enzyme system generating these reactive-oxygen species is believed to be an NADPH oxidase located in the plasma membrane and sharing similarities with the NADPH oxidase from mammalian macrophages. Antibodies directed against individual subunits (p22 phox , p47 phox , p67 phox ) of the human NADPH oxidase cross-react with soybean proteins of a similar size and subcellular location. An extensive expression screening of a soybean cDNA-library with the anti-human NADPH oxidase antibodies gave a single class of cDNA-clones for each antibody. However, the sequence analysis of these clones clearly demonstrates that the different antibodies recognise proteins which are unrelated to the expected oxidase subunits. The anti-p22 phox antibody recognised a microsomal protein with no significant homology to any known protein in the database. One anti-p47 phox antibody cross-reacted with the UDP-glucose dehydrogenase and another antibody bound to the chaperon peptidyl prolyl-cis-trans isomerase, both soluble cytosolic proteins. The anti-p67 phox antibody detected the soluble enzyme acetohydroxy acid reductoisomerase. Chromatography of soybean protein extracts on an ion-exchange column (MonoQ, FPLC) gave a perfect comigration of the enzyme activity with the antibody signal, thus confirming these unexpected results by independent biochemical experiments.

NADH oxidase activity of soybean plasma membranes inhibited by submicromolar concentrations of ATP.

The activity of an auxin-stimulated NADH oxidase activity from soybean hypocotyls was inhibited by submicromolar concentrations of ATP. Auxins are plant growth regulators that increase the rate of cell enlargement in plant stems. A synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), was used. The inhibition was half maximal at 1 nM ATP and was not observed with other nucleotides and nucleosides. The inhibition was the result of an increase in the Km for NADH from about 60 microM to > 100 microM and was noncompetitive. The decrease in Km due to ATP was enhanced by the addition of 1 microM 2,4-D. The Vmax of the plasma membrane NADH oxidase was approximately doubled (1.5-2.8-fold) by ATP and by 1 microM 2,4-D. No further increase in the Vmax was observed by the combination of 1 nM to 0.1 mM ATP in the presence of 1 microM 2,4-D. The results demonstrate a response of the NADH oxidase activity of isolated vesicles of soybean plasma membranes to ATP distinct from that observed previously with other nucleotide di- and triphosphates. The results are suggestive either of control of the cell surface NADH oxidase by phosphorylation or a direct response to ATP binding at nanomolar concentrations of ATP.

NADH oxidase activity present on both the external and internal surfaces of soybean plasma membranes

Soybean plasma membranes exhibit NADH oxidase activities accessible to NADH supplied to either the external or internal membrane surfaces. Activity at the external surface was demonstrated using plasma membranes of right side-out orientation prepared by aqueous two-phase partition and using intact soybean cells grown in culture. Activity at the internal membrane surface was demonstrated using vesicles of inside-out orientation obtained by preparative free-flow electrophoresis or by aqueous two-phase partition following freeze–thaw or Brij detergent treatment to invert some of the right side-out vesicles. The NADH oxidase activity of both membrane surfaces appeared to be stimulated by the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D). Following the freeze–thaw treatment utilized to evert the vesicles, however, the NADH oxidase activity of right side-out vesicles was greatly reduced. A 2,4-D-stimulated activity of these vesicles could be restored by treatment first with reduced glutathione and then with hydrogen peroxide or oxidized glutathione. Upon treatment with 0.1% Triton X-100, both right side-out and inside-out vesicles exhibited approximately the same specific activity and the same 2,4-D-stimulated component of activity equal to the sum of the activities of the right side-out and inside-out vesicles assayed separately. These findings suggest that a significant NADH oxidase activity of the soybean plasma membrane responsive to 2,4-D resides on the external plasma membrane surface. This latter activity is unlikely to function under physiological conditions in the direct oxidation of NADH and alternative functions, such as in protein disulfide–thiol interchange, would be more appropriate.

Chlorsulfuron blocks 2,4-D-induced cell enlargement and NADH oxidase in excised sections of soybean hypocotyls

Elongation of soybean hypocotyl sections induced by the auxin herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), was blocked by micromolar concentrations of the sulfonylurea herbicide chlorsulfuron (2-chloro- N-[[(4-methoxy-6-methyl-1,3,5-trizin-2yl)amino]carbonyl]benzenesulfonamide). The inhibition at equimolar concentrations of 2,4-D and chlorsulfuron was overcome by a 10- or 100-fold excess of 2,4-D. Stimulation by 2,4-D of an 2,4-D-responsive NADH-oxidase activity of the soybean plasma membrane also was blocked by the presence of concentrations of chlorsulfuron equimolar to the 2,4-D. Chlorsulfuron alone was largely without effect on either cell elongation or the auxin-stimulated NADH-oxidase activity over a range of chlorsulfuron concentrations. The results show a clear correlation between inhibition of auxin-stimulated cell elongation and the inhibition of an auxin-stimulated component of a plasma membrane NADH-oxidase activity.