Prachuab Kwanyuen

Isolation and purification of diacylglycerol acyltransferase from germinating soybean cotyledons

Abstract A homogeneous preparation of the enzyme that catalyses triacylglycerol synthesis has been achieved for the first time from plant tissue. Diacylglycerol acyltransferase (EC 2.3.1.20) was solubilized from purified microsomes prepared from germinating soybean (Glycine max L. Merr. cv. Dare) cotyledons with the non denaturing zwitterionic detergent, CHAPS. The enzyme was isolated from CHAPS solubilized microsomal proteins and purified by Sepharose CL-4B chromatography and agarose gel electrophoresis. Purified diacylglycerol acyltransferase exhibited a single Schlieren image during analytical ultracentrifugation. The homogeneity of the enzyme was confirmed by high-pressure liquid chromatography using gel filtration columns. The purified enzyme was devoid of lipase (EC 3.1.1.3), glycerolphosphate acyltransferase (EC 2.3.1.15), lysolecithin acyltransferase (EC 2.3.1.23), acylglycerol acyltransferase (EC 2.3.1.22), and 1-acylglycerolphosphate acyltransferase (EC 2.3.1.51) activities. Purified diacylglycerol acyltransferase activity was stable for three months at −20°C. Hyperbolic enzyme kinetics were observed using sn-1,2-diolein and stearoyl-CoA or oleoyl-CoA as substrates. The apparent Km using sn-1,2-diolein was 60 μM with stearoyl-CoA, and 113 μM with oleoyl-CoA. This report has documented a method by which highly purified diacylglycerol acyltransferase may be obtained from germinating soybean cotyledons.

Triacylglycerol synthesis and metabolism in germinating soybean cotyledons

Abstract The objective of this study was to investigate the ability of germinating soybean (Glycine max L. Merr. cv. Dare) cotyledons to synthesize triacylglycerol, for the purpose of identifying an alternative tissue source for the isolation and purification of the enzyme diacylglycerol acyltransferase (EC 2.3.1.20). Aspects of glycerolipid metabolism were examined in etiolated cotyledons and greening cotyledons with intact or detached epicotyls at various stages of germination. Regardless of tissue morphology or age, cotyledons from all treatments were found to be capable of triacylglycerol synthesis from [2-14C]acetate. In general, the rate of triacylglycerol synthesis in etiolated cotyledons was greater than in greening cotyledons with intact epicotyls. The greatest triacylglycerol synthetic activity (2.1 nmol 14C/h per g dry weight), however, was attained at 20 days after germination in greening cotyledons with detached epicotyls. That rate was approximately one-half of the maximal rate of triacylglycerol synthesis from [2-14C]acetate reported for developing Dare seed. Furthermore, it was shown that the de novo synthesized triacylglycerol in germinating soybean cotyledons exhibited zero-order metabolic kinetics. Hence these data indicated a subcellular compartmentation of the synthetic and hydrolytic mechanisms associated with triacylglycerol metabolism in germinating soybean cotyledons. The conclusion drawn from these findings was that germinating soybean cotyledons would be a suitable medium for the isolation, purification, and subcellular localization of diacylglycerol acyltransferase, the enzyme which catalyses triacylglycerol synthesis.

Subunit and amino acid composition of diacylglycerol acyltransferase from germinating soybean cotyledons.

The subunit and amino acid composition of the enzyme that catalyses triacylglycerol synthesis was determined for the first time from plant tissues. Diacylglycerol acyltransferase (acyl-CoA:1,2-diacylglycerol O-acyltransferase, EC 2.3.1.20) purified from germinating soybean (Glycine max L. Merr. cv. Dare) cotyledons, dissociated into three nonidentical subunits having apparent molecular masses of 40.8, 28.7, and 24.5 kDa. The respective subunits occurred in a 1:2:2 molar ratio in the native enzyme. Five peptides in that molar ratio were assumed to constitute a monomer having a putative molecular mass of 153.1 kDa. Based upon the apparent molecular mass of purified diacylglycerol acyltransferase after delipidation (1539 kDa), there was a high probability that the complete structure of the native enzyme from soybean contained ten identical monomers. The polarity index of each subunit was less than 21%, far below the 40% boundary reported for membrane bound proteins. Hydrophobic amino acids accounted for greater than 48% of the composition in each subunit. It was predicted from these data that the native enzyme contained 12,525 amino acid residues, and that the two smaller subunits were more deeply embedded in the membrane than the 40.8 kDa subunit. Attempts to reactivate the denatured or delipidated protein were not successful.

Non-antibiotic selection systems for soybean somatic embryos: the lysine analog aminoethyl-cysteine as a selection agent

BackgroundIn soybean somatic embryo transformation, the standard selection agent currently used is hygromycin. It may be preferable to avoid use of antibiotic resistance genes in foods. The objective of these experiments was to develop a selection system for producing transgenic soybean somatic embryos without the use of antibiotics such as hygromycin.ResultsWhen tested against different alternate selection agents our studies show that 0.16 μg/mL glufosinate, 40 mg/L isopropylamine-glyphosate, 0.5 mg/mL (S-(2 aminoethyl)-L-cysteine) (AEC) and the acetolactate synthase (ALS) inhibitors Exceed® and Synchrony® both at 150 μg/mL inhibited soybean somatic embryo growth. Even at the concentration of 2 mg/mL, lysine+threonine (LT) were poor selection agents. The use of AEC may be preferable since it is a natural compound. Unlike the plant enzyme, dihydrodipicolinate synthase (DHPS) from E. coli is not feed-back inhibited by physiological concentrations of lysine. The dapA gene which codes for E. coli DHPS was expressed in soybean somatic embryos under the control of the CaMV 35S promoter. Following introduction of the construct into embryogenic tissue of soybean, transgenic events were recovered by incubating the tissue in liquid medium containing AEC at a concentration of 5 mM. Only transgenic soybeans were able to grow at this concentration of AEC; no escapes were observed.ConclusionGenetically engineered soybeans expressing a lysine insensitive DHPS gene can be selected with the non-antibiotic selection agent AEC. We also report here the inhibitory effects of glufosinate, (isopropylamine-glyphosate) (Roundup®), AEC and the ALS inhibitors Exceed® and Synchrony® against different tissues of soybean

Seed Nitrogen Mobilization in Soybean: Effects of Seed Nitrogen Content and External Nitrogen Fertility

ABSTRACT Soybean breeding programs have developed genetic lines with relatively low seed protein, which could negatively impact early seedling growth in low fertility conditions commonly encountered in the field. In these experiments, seed protein mobilization and its regulation in situ in soybean lines with different seed protein levels was investigated. The results showed that rates of nitrogen (N) release from cotyledons were much lower with decreasing levels of N in seed. Patterns of proteolysis of the storage proteins glycinin and β -conglycinin and their subunits were not different, but breakdown rates were slower. Seed N release rates increased somewhat when external N was supplied to roots of the developing seedlings, suggesting the involvement of source/sink controls. The effect appeared to be down-stream from proteolysis, as rates of protein breakdown were not altered. The results indicate that low seed protein levels will lead to reduced seedling fitness in low fertility soil conditions unless fertilizer N is applied.

Role of Diacylglycerol Acyltransferase in Regulating Oil Content and Composition in Soybean

Accessions of the USDA Soybean ( Glycine max L. Merr.) Germplasm Collection exhibit genetic diversity for oil concentration ranging from 12 to 27 percent of dry weight. Although oil concentration is a highly heritable quantitative genetic trait, the genetic and biochemical basis for genotypic differences in the oil content of soybean seed is unknown. Recent knowledge on biological regulation of this trait has emerged from research on diacylglycerol acyltransferase (EC 2.3.1.20), the enzyme that catalyses triacylglycerol synthesis. Diacylglycerol acyltransferase purified from the cv. Dare has a native mass of about 1.5 MDa. Structural analysis suggests the protein consists of 10 monomers having three nonidentical subunits in a 1:2:2 molar ratio. Kinetics of the enzyme purified from soybeans exhibiting high or low oil content suggest that genotypic differences in oil content may be governed by gene dosage effects. However, subtle conformational changes in protein structure may influence oil composition, as evidenced by apparent substrate specificities observed in germplasm containing low-palmitic acid. Therefore, diacylglycerol acyltransferase may play a unique role in determining the content and composition of triacylglycerol in soybean.