Genomic regions governing soybean seed nitrogen accumulation

Nitrogen accumulation in the form of seed protein takes place in soybean [Glycine max (L.) Merr.] during the reproductive stages of development. The purpose of this study was to relate genotypic differences in seed nitrogen accumulation with genomic regions controlling nitrogen accumulation in soybean during R₅, R₆, and R₇ growth stages. A population of 101 F_6∶8 recombinant inbred lines (RIL) developed from a cross of N87-984-16×TN93-99 was utilized. The RIL were grown at the University of Tennessee, Knoxville Experiment Station, in a randomized complete block design with three replications in 2002. Seed nitrogen was determined from pod samples harvested at the R₅, R₆, and R₇ growth stages. A significant (P<0.05) difference among genotypes was found for nitrogen accumulation at all three growth stages. Single-factor ANOVA revealed that quantitative trait loci (QTL) governing nitrogen accumulation in soybean seed were distributed in the linkage groups A2, B2, D1a, D1b, E, G, and M. Phenotypic variation explained by an individual QTL ranged from 5 to 11.6%. These QTL may provide useful marker-assisted selection opportunities for soybean protein improvement.     

Restriction fragment length polymorphism analysis of soybean fatty acid content

The quality of soybean [Glycine max (L.) Merr.] oil depends greatly upon its fatty acid composition. The objective of this study was to map quantitative trait loci affecting the relative content of the fatty acids composing soybean seed oil. The mapping was done in a population formed from a cross between aG. max experimental line and aG. soja plant introduction. Sixty F2-derived lines from this population were genotyped with 243 restriction fragment length polymorphism, five isozyme, one storage protein and three morphological markers. The genotyped lines were grown in a replicated trial, and the seed harvested was analyzed for palmitic, stearic, oleic, linoleic and linolenic fatty acids. Markers significantly (P>0.005) associated with each fatty acid were found. Individual markers were associated with up to 38% of the total variance in specific fatty acids among the F2 lines. The greatest associations between markers and fatty acid content were observed with markers on two major linkage groups. Joint contribution of North Central Region, USDA-ARS and Journal Paper no. J-14849 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011; Project 2974.     

Using hydrophilic polymers to improve uptake of manganese fertilizers by soybeans

Manganese deficiency in soybeans (Glycine max (L.) Merr.) is a common problem in many parts of the world. Recent research has demonstrated that the addition of gel-forming hydrophilic polymers with plant nutrients may enhance the availability and effectiveness of some soil-applied nutrients. This greenhouse study was designed to determine if the addition of hydrated cross-linked polyacrylamide polymers could increase plant recovery of commonly used Mn fertilizers by soybeans. Four Mn sources (MnO, MnSO₄·4H₂O, MnCl₂, and MnEDTA) were band applied at two concentrations to a low-Mn soil with and without one of two polymers. Addition of either polymer alone or MnO had no effect on leaf or stem Mn concentration, but when MnSO₄·4H₂O, MnCl₂, or MnEDTA were added with a polymer, leaf Mn accumulation swere increased an average of 89%, compared with those Mn sources applied alone. Plant accumulation of Mn from MnO was no greater than the control treatment and uptake was not increased following the addition of polymer. At the conclusion of the experiment, the polymers were still hydrated and the fertilizer band contained an abundance of roots. The use of a hydrophilic polymer with soluble Mn fertilizers appears to enhance the recovery by plants and may lead to lower Mn application rates or perhaps less frequent applications.     

Sucrose fatty acid esters enhance efficiency of foliar-applied urea-nitrogen to soybeans

A greenhouse study is described showing the effect of sucrose fatty acid esters (SFE) applied to soybeans (Glycine max (L.) Merrill cv Fukuyutaka) during the flowering and/or pod-filling periods on the efficiency of foliar-applied urea-nitrogen. SFE applied in combination with urea delayed senescence and when applied during both the flowering and pod-filling periods increased seed yields by 103% and nitrogen accumulation by 132% as compared to urea alone. Average total recovery of¹⁵N-urea in the above ground portions of the plant was 20.6%. SFE combined with urea increased the average recovery to 34.8%. Recovered¹⁵N-urea was only a small portion of the total nitrogen content of the plant. The yield increase resulting from a foliar application of urea may have been due to the leaves continuing to export photosynthates to the nodules hence maintaining the nodules' nitrogen fixing activity for a longer period of time. The addition of SFE to the urea solution increased the retention and/or absorption of urea and increased translocation of urea-nitrogen to the seeds.     

Response of promiscuously nodulating soybean to N and P fertilization andBradyrhizobium inoculation in a ferruginous tropical soil (Haplustalf)

Field trials were conducted at Samaru, Nigeria over a three-year period (1986–88) to study the effects of N and P fertilization on the response of promiscuously nodulating soybean toBradyrhizobium japonicum inoculation in a ferruginous tropical soil. Phosphorus fertilization enhanced nodulation, while N fertilization had no consistent effect on nodulation. Both N and P increased dry matter production. Seed yields were not influenced by the application of N. However, P increased seed yields in two out of three years. Response of seed yield to P fertilization was significant up to 26.4 kg P ha^–1.Bradyrhizobium inoculation consistently enhanced nodulation while it increased seed yield in only one out of three years. Results demonstrate that P is an important nutrient for soybean production in ferruginous tropical soils. The roles of promiscuously nodulating soybean in the maintenance of tropical soil fertility are discussed.     

Genetic regulation of linolenic acid concentration in wild soybean Glycine soja accessions

Soybean [Glycine max (L.) Merr.] oil from current commercial cultivars typically contains ca. 8% linolenic acid. Inheritance studies have shown that linolenic acid concentration in soybean seed is determined by at least two genes which govern activity of the predominant ω-6 and ω-3 desaturases. Selection of germplasm exhibiting homozygous recessive alleles that encode these desaturases has enabled development of soybeans having less than 3.0% linolenic acid. However, accessions of the wild ancestor of modern soybean cultivars, Glycine soja (Sieb. and Zucc.), have oils containing twice the highest linolenic acid concentration found in normal G. max cultivars. Although little is known about inheritance of linolenic acid in wild soybean, it would appear that additional or alternative forms of genes may govern its synthesis. To test this hypothesis, cultivated soybean germplasm was hybridized with wild soybean genotypes having significant differences in linolenic acid concentration. Seed of F₃ progeny from these G. max x G. soja populations exhibited distinct segregation patterns for relative estimates of ω-6 and ω-3 desaturase activity. Frequency class distribution analyses of the segregation patterns, and linear relations between median ω-6 or ω-3 desaturation estimates and corresponding linolenic acid concentration among allelic classes from these populations suggested the high-linolenic acid trait in wild soybean genotypes was determined by a set of desaturase alleles that were different from corresponding alleles in G. max. Introgression of these alternative alleles in G. max germplasm opens a new avenue of research on the genitic regulation of linolenic acid, and may lead to the production of highly polyunsaturated soybean oils for various industrial applications.     

Relationship between seed mass and linolenic acid in progeny of crosses between cultivated and wild soybean

Soybean [Glycine max (L.) Merr.] oil from current commercial cultivars typically contains ca. 8% linolenic acid (18:3). Applications of plant biotechnology have enabled plant breeders to develop germplasm having as low as 2.0% 18:3. Oils that are naturally low in 18:3 exhibited improved flavor characteristics and greater oxidative stability in high-temperature frying applications compared to hydrogenated soybean oil. As an extension of that research, efforts are underway to characterize genes in soybean that govern expression of higher than normal 18:3 concentration. Such oils may be of interest to the oleochemicals industry for various nonfood applications. Relatively high 18:3 in seed oil is a characteristic trait of the ancestor of modern soybean cultivars, Glycine soja (Sieb. and Zucc.). Accessions of this species have rarely been utilized in soybean improvement, and thus represent a virtually untapped genetic resource for genes governing 18:3 synthesis. We have hybridized cultivated soybean with wild soybean plant introductions. F_3:4 seed from the resultant G. max × G. soja populations exhibited a wide segregation pattern for 18:3 and seed mass. A strong negative association was found between 18:3 concentration and seed mass. Oil concentration was positively correlated with seed mass. Evaluation of glycerolipid composition revealed that high 18:3 was not associated with an altered proportion of phospholipid and triacylglycerol among lines segregating for seed mass. Thus, smaller seed mass may be a convenient trait to distinguish future soybean cultivars with highly polyunsaturated oils from other cultivars in production.     

Use of NMR for predicting protein concentration in soybean seeds based on oil measurements

Increasing the level of protein in soybean seeds has been a major target for soybean [Glycine max (L.) Merr.] breeders. The objective of this study was to examine the potential of predicting soybean seed protein based on oil values as determined by NMR. Seed protein and oil concentrations were determined in an F₂ population generated from the cross between a G. max (NK, S08-80), and a G. soja (PI 458536) cultivar. The protein concentration in the population ranged from 40.4 to 52.6%. Protein-oil regression analysis was used to generate an equation for predicting seed protein concentration based on oil readings. The regression equation Protein=62.3–1.3 [Oil] (R ²=0.46) was developed, with a corresponding correlation of −0.69 between the traits. With this equation, the mean protein concentration of the selected 25% of the population (a simulated breeding pressure) was greater than the mean of the unselected population (46.1%, SE=0.13) by about 1.9%. Individual F₂ plants that exceeded the mean protein value of the population constituted 86.4% of the selected samples. Selection based on oil concentration, however, failed to include 27.1% of the plants that were among the top 25% for protein concentration. Selection of high-protein plants based on NMR oil measurement was reasonably effective in the test population and might offer a new and rapid method of selecting high-protein individuals in soybean populations derived from the wild soybean progenitor, G. soja. If further tested on other populations and samples, it might be used as an analytical alternative for an indirect measurement of protein concentration based on NMR measurements of the oil.     

Optimization of coomassie staining for quantitative densitometry of soybean storage proteins in gradient gel electrophoresis

Soybean (Glycine maxL. Merr., cv. Dare) protein subunits were separated by gradient gel electrophoresis and analyzed by two-dimensional densitometry with computer-aided volume integration. Significant differences in the time required to achieve equilibrium staining with Coomassie Blue were revealed among the various polypeptides. Bands corresponding to lipoxygenase reached staining equilibrium in 2.7 h, whereas longer periods were required for polypeptides of β-conglycinin (5.5 to 6.7 h) and of glycinin (8.6 to 9.2 h). These differences among polypeptides could be attributed in part to changes in gradient concentration within the polyacrylamide gel. Optimal staining intensity among all soluble proteins extracted from soybean seed was reached after staining for 8 h. Shorter than optimal staining times lead to significant underestimation of parameters such as the percentage of β-conglycinin and glycinin of total soluble protein.     

Over-Expression of GmGIa-Regulated Soybean miR172a Confers Early Flowering in Transgenic Arabidopsis thaliana

Flowering is a pivotal event in the life cycle of plants. miR172 has been widely confirmed to play critical roles in flowering time control by regulating its target gene expression in Arabidopsis. However, the role of its counterpart in soybean remains largely unclear. In the present study, we found that the gma-miR172a was regulated by a GIGANTEA ortholog, GmGIa, in soybean through miRNA metabolism. The expression analysis revealed that gma-miR172a has a pattern of diurnal rhythm expression and its abundance increased rapidly as plants grew until the initiation of flowering phase in soybean. One target gene of gma-miR172a, Glyma03g33470, was predicted and verified using a modified RLM 5′-RACE (RNA ligase-mediated rapid amplification of 5′ cDNA ends) assay. Overexpression of gma-miR172a exhibited an early flowering phenotype and the expression of FT, AP1 and LFY were simultaneously increased in gma-miR172a-transgenic Arabidopsis plants, suggesting that the early flowering phenotype was associated with up-regulation of these genes. The overexpression of the gma-miR172a-resistant version of Glyma03g33470 weakened early flowering phenotype in the toe1 mutant of Arabidopsis. Taken together, our results suggested that gma-miR172a played an important role in GmGIa-mediated flowering by repressing Glyma03g33470, which in turn increased the expression of FT, AP1 and LFY to promote flowering in soybean.     

Reevaluation of grading standards and discounts for fungus-damaged soybean seeds

The proportion of off-colored seed is a primary quality-rating factor that influences the market grade of soybean,Glycine max L. Merrill. Off-colors, attributed to biological agents such as fungi and viruses, are visual characteristics that arbitrarily result in lower grade ratings when soybeans show more than ten percent discoloration. These damaged seeds may be further classified under current United States Department of Agriculture, Federal Grain Inspection Service standards as: moldy, weathered, bicolored, mottled, or other colors. Presently, all types of fungal damage and virus symptoms are evaluated equally in the application of discount schedules at grain-elevators. However, recent information shows that superficial discoloration caused by some biological agents does not lower grain quality. Indeed, methods are available to distinguish those symptoms that actually reduce seed quality from those that are more cosmetic in nature. This communication provides guidance on fungi that are involved most often in seed damage or discoloration. The following organisms are listed in descending order of importance:Phomopsis longicolla, Alternaria, Nematospora coryli, Fusarium graminearum, Colletotrichum, andCercospora kikuchii. Discoloration caused byPeronospora manchurica (downy mildew) and the soybean mosaic virus have limited effect on seed quality. Discoloration caused by each of these agents may be identified by recognizable characteristics. Therefore, knowledge of the relative impact of these organisms on seed quality should be practiced in the application of soybean grading standards and discount schedules.     

Oxidation of acylglycerols and phosphoglycerides by soybean lipoxygenase

Lipoxygenase (EC 1.13.11.12) catalyzes the incorporation of oxygen into polyunsaturated fatty acids, resulting in the formation of their corresponding hydroperoxides. The ability of a commercial preparation of soybean (Glycine max L. Merr.) lipoxygenase to catalyze the oxidation of acylglycerols and phosphoglycerides was investigated. The oxidation rate of trilinolein increased nearly 100% when 5 mM deoxycholate was added to the reaction medium. With further increases in the concentration of deoxycholate, the oxidation rate decreased slightly. The pH profile of trilinolein oxidation was bell-shaped. The rate of oxidation was maximal at pH 8, and it decreased to near zero at pH 5 and pH 11. Even under optimal conditions, the rate of trilinolein oxidation was only 3% of that of linoleic acid, and analysis of time course data showed that, at most, 15% of available linoleate was oxidized. In contrast to the slow rate of trilinolein oxidation, tested phosphoglycerides and diacylglycerols were oxidized at moderate rates. The rate of phosphoglyceride oxidation depended upon the structure of the polar head group and varied between 7–28% of the rate of linoleic acid oxidation. Diacylglycerols reacted at a rate that was 40% of that of linoleic acid. Analysis of the time course of 1,3-dilinolein oxidation showed that as much as 67% of the available linoleate could be converted to the corresponding hydroperoxide. Analyses by high-performance liquid chromatography revealed that more than 20% of the 1,3-dilinolein was converted to unidentified products that are not hydroperoxides.     

Induced resistance in soybean toHelicoverpa zea: Role of plant protein quality

Resistance in soybean toHelicoverpa zea is comprised of both constitutive and inducible factors. In this study, we investigated the induction of resistance byH. zea in both greenhouse and field studies. In a greenhouse experiment, fourth-instarH. zea growth rates were reduced by 39% after 24 hr feeding and by 27% after 48 hr when larvae fed on previously wounded V3 foliage (cv. Forrest) compared with undamaged foliage. In a field study, the weight gain by larvae was more than 52% greater when larvae fed for 72 hr on undamaged R2/R3 soybean plants (cv. Braxton) compared to those that fed on previously wounded plants. A significant component of the induced resistance is due to a decline in the nutritional quality of foliar protein following foliar damage byH. zea. Foliar protein was extracted from damaged and undamaged foliage and incorporated into artificial diets. Larval growth was reduced 26% after four days and 49% after seven days on diets containing protein from damaged plants compared to larvae feeding on foliar protein from undamaged plants. Chemical analyses of protein quality also indicated a decline in quality in damaged plants compared to unwounded plants. Increases in lipoxygenase activity (53%), lipid peroxidation products (20%), and trypsin inhibitor content (34%) were observed in protein from wounded plants. Moreover, a 5.9% loss in free amines and 19% loss in total thiols occurred in protein from wounded plants. Larval feeding causes a significant increase in foliar lipoxygenase activity that varied among genotypes. Lipoxygenase isozymes were measured at pH 5.5, pH 7.0, and pH 8.5 in V3 stage plants of Forrest, Hark, D75-1069, and PI 417061 genotypes. Lipoxygenase activity in each genotype was significantly increased after 72 hr of larval feeding at each pH level tested, with the exception of lipoxygenase isozymes at pH 5.5 in genotype PI 417061. Larval feeding on R2/R3 stage plants (field-grown cv. Braxton) for six days also increased foliar lipoxygenase activity.     

Effects of benzoic and cinnamic acids on growth,mineral composition,and chlorophyll content of soybean

Organic acids are major water-soluble allelochemicals found in soil infested with quackgrass and are involved in several processes that are important in plant growth and development. This study was carried out to gain more information on the effects of benzoic acid (BEN) andtrans-cinnamic acid (CIN) on growth, mineral composition, and chlorophyll content of soybean [Glycine max (L.) Merr. cv. Maple Bell] grown in nutrient solution. The two allelochemicals reduced root and shoot dry biomass of soybean. Treated plants had fewer lateral roots and tended to grow more horizontally compared to the untreated plants. Lateral roots were stunted and less flexible. The amounts of P, K, Mg, Mn, Cl^–, and SO ₄ ^2– were lower, and Zn and Fe contents were higher in roots of plants grown with BEN or CIN as compared to untreated plants. Shoots of plants grown with the allelochemical showed greater accumulation of Ca, Mg, and Zn, whereas P and Fe contents were reduced. The BEN and CIN also caused reductions in leaf chlorophyll content. The BEN and CIN may be responsible for negative allelopathic effects of quackgrass on soybean by inhibiting root growth, by altering ion uptake and transport, and by reducing chlorophyll content.Contribution 493 of the Soils and Crop Research Center.     

Identification of Cold-Responsive miRNAs and Their Target Genes in Nitrogen-Fixing Nodules of Soybean

As a warm climate species, soybean is highly sensitive to chilling temperatures. Exposure to chilling temperatures causes a significant reduction in the nitrogen fixation rate in soybean plants and subsequent yield loss. However, the molecular basis for the sensitivity of soybean to chilling is poorly understood. In this study, we identified cold-responsive miRNAs in nitrogen-fixing nodules of soybean. Upon chilling, the expression of gma-miR397a, gma-miR166u and gma-miR171p was greatly upregulated, whereas the expression of gma-miR169c, gma-miR159b, gma-miR319a/b and gma-miR5559 was significantly decreased. The target genes of these miRNAs were predicted and validated using 5'' complementary DNA ends (5''-RACE) experiments, and qPCR analysis identified putative genes targeted by the cold-responsive miRNAs in response to chilling temperatures. Taken together, our results reveal that miRNAs may be involved in the protective mechanism against chilling injury in mature nodules of soybean.     

Phenolic acid content of soils from wheat-no till,wheat-conventional till,and fallow-conventional till soybean cropping systems

Soil core (0–2.5 and/or 0–10 cm) samples were taken from wheat no till, wheat-conventional till, and fallow-conventional till soybean cropping systems from July to October of 1989 and extracted with water in an autoclave. The soil extracts were analyzed for seven common phenolic acids (p-coumaric, vanillic,p-hydroxybenzoic, syringic, caffeic, ferulic, and sinapic; in order of importance) by high-performance liquid chromatography. The highest concentration observed was 4 g/g soil forp-coumaric acid. Folin & Ciocalteu's phenol reagent was used to determine total phenolic acid content. Total phenolic acid content of 0- to 2.5-cm core samples was approximately 34% higher than that of the 0- to 10-cm core samples. Phenolic acid content of 0- to 2.5-cm core samples from wheat-no till systems was significantly higher than those from all other cropping systems. Individual phenolic acids and total phenolic acid content of soils were highly correlated. The last two observations were confirmed by principal component analysis. The concentrations were confirmed by principal component analysis, tions of individual phenolic acids extracted from soil samples were related to soil pH, water content of soil samples, total soil carbon, and total soil nitrogen. Indirect evidence suggested that phenolic acids recovered by the water-autoclave procedure used came primarily from bound forms in the soil samples.The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of products named, nor criticism of similar ones not mentioned.     

Temperature and cultivar effects on soybean seed oil and protein concentrations

The soybean [Glycine max (L.) Merr.] industry is interested in cultivar and climate effects on seed composition. These factors may underlie the known geographic variation in seed protein and oil concentrations. Regression analyses were used to test hypotheses of the effect of temperature and cultivar on oil and protein concentrations of soybean seed using a large data set from the U.S.A. Soybean Uniform Tests. The data set included 20 cultivars representing 10 maturity groups across 60 locations (latitude 29.4 to 47.5° N) for a total of 1863 cultivar by location by year observations. Temperature was determined for each observation as the average daily mean temperature from predicted first pod (first pod at least 5 mm long), using the SOYGRO phenology model, to observed maturity. The mean temperature ranged from 14.6 to 28.7°C among the observations. Linear, quadratic, and linear plateau regression models of oil and protein concentrations vs. temperature were evaluated. The quadratic model gave the best-adjusted R ² values for oil and protein with temperature, of 0.239 and 0.003, respectively. The analyses showed that the oil concentration increased with increasing temperature and approached a maximum at a mean temperature of 28°C. Unaccounted variation in the protein concentration may be from other factors such as photoperiod, water stress, or high temperatures during seed fill. Protein plus oil had a linear relationship with temperature (adjusted partial R ²=0.183). These data document the contribution of climate and cultivar to geographic variability of oil and protein concentrations in the United States.     

Relation between diacylglycerol acyltransferase activity and oil concentration in soybean

Diacylglycerol acyltransferase (EC 2.3.1.20; DGAT) catalyzes synthesis of triacylglycerol from acyl-CoA and diacylglycerol. Activity of this enzyme and developmental changes in oil accumulation were estimated at various stages of seed growth in soybean germplasm with phenotypic differences in oil content. Oil deposition in seed of these genotypes followed a sigmoid pattern that was modeled to predict incremental rates of oil accumulation during seed development. A strong positive correlation was found between the estimated peak rate of oil deposition (near the mid-term of seed development) and oil concentration in mature seed. At saturating substrate levels, DGAT activity measured near the peak rate of oil deposition also was correlated positively with oil phenotype. In the latter stages of seed development, a positive correlation between estimates of enzyme activity at or below the apparent K _m for diolein and comparable oil accumulation rates was attributed to reduced synthesis of substrates and/or potential change in affinity for substrate as suggested by an increase in apparent K _m for diolein in older seed. These data indicated that DGAT activity may be a rate-limiting step in triacylglycerol synthesis. However, it is difficult to accept the idea of a single rate-limiting step at the end of a complex metabolic pathway. Because oil is a quantitatively inherited trait, several genes determine genotypic differences in oil content among soybeans. Hence, DGAT activity may be an indicator of coordinated genetic expression of gene-products in the entire glycerolipid synthetic pathway for a given genotype. In any case, results of this investigation demonstrated that genotypic differences in DGAT activity contributed to expression of genetic variation in oil content among soybean gemplasm.     

Effects of salicylic acid on plant-water relationships

Soybean seedlings [Glycine max (L.) Merr.] were used as the test species to study the allelopathic influence of salicylic acid (SA) on short- and long-term plant water status. Plants were grown in greenhouse conditions in nutrient culture medium amended with SA. Treatments were initiated 10 days after germination and continued for either 14 or 28 days. The threshold for inhibition of seedling growth over a 28-day treatment was 0.15 mM SA. Seedlings grown with 0.3 mM SA consistently had higher leaf diffusive resistance and lower transpiration and water potentials than control plants. The stable carbon isotope ratio (¹³C:¹²C) in tissue from both the 0.15 and 0.30 mM SA-treated plants was significantly higher than control seedlings, indicating SA caused a chronic water stress during the 28-day treatment. These data show that an interference with plant-water relationships is one mechanism whereby this allelochemical inhibits plant growth.This study was supported by National Science Foundation grant STI-8902066.     

Soybean lipoxygenase-promoted oxidation of free and esterified linoleic acid in the presence of deoxycholate

Lipoxygenase (EC 1.13.11.12) catalyzes the reaction between oxygen and polyunsaturated fatty acids to give fatty acid hydroperoxides. Recent work showed that soybean lipoxygenase 1 can oxidize diacylglycerols when deoxycholate is present in the reaction medium. Conditions were sought to maximize 1,3-dilinolein oxidation with a commercial soybean lipoxygenase preparation. It was found that dilinolein was oxidized most rapidly in a multicomponent buffer medium that contained 10 mM deoxycholate between pH 8 and 9. When dilinolein oxidation was conducted in the individual components of the multicomponent buffer, the oxidation rate decreased two- to threefold. Addition of 0.2 M NaCl to one of the components, Tricine buffer, caused a twofold increase in the oxidation rate, demonstrating that high ionic strength is a major factor promoting rapid oxidation in the multicomponent buffer. In the deoxycholate multicomponent buffer, the order of reactivity toward oxidation was monolinolein>methyl linoleate≈ linoleic acid>dilinolein. Competition experiments in which mixtures of the substrates were presented simultaneously to lipoxygenase in the presence of deoxycholate showed that linoleic acid was the most reactive substrate. When no surfactant was present or when the surfactant was Tween 20, linoleic acid was the most rapidly oxidized substrate. Overall, the results demonstrate that monolinolein and methyl linoleate are just as reactive, or more so, as linoleic acid to oxidation by lipoxygenase under specified reaction conditions. In competition experiments, linoleic acid oxidation predominates, probably because its free carboxyl functionality allows it to be preferentially bound to the active site of lipoxygenase.