Thomas A. McKeon

The effect of plant-hormone pretreatments on ethylene production and synthesis of 1-aminocyclopropane-1-carboxylic acid in water-stressed wheat leaves

Excised wheat (Triticum aestivum L.) leaves, when subjected to drought stress, increased ethylene production as a result of an increased synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) and an increased activity of the ethyleneforming enzyme (EFE), which catalyzes the conversion of ACC to ethylene. The rise in EFE activity was maximal within 2 h after the stress period, while rehydration to relieve water stress reduced EFE activity within 3 h to levels similar to those in nonstressed tissue. Pretreatment of the leaves with benzyladenine or indole-3-acetic acid prior to water stress caused further increase in ethylene production and in endogenous ACC level. Conversely, pretreatment of wheat leaves with abscisic acid reduced ethylene production to levels produced by nonstressed leaves; this reduction in ethylene production was accompanied by a decrease in ACC content. However, none of these hormone pretreatments significantly affected the EFE level in stressed or nonstressed leaves. These data indicate that the plant hormones participate in regulation of water-stress ethylene production primarily by modulating the level of ACC.

Cloning and characterization of a cDNA encoding diacylglycerol acyltransferase from castor bean

The oil from castor seed (Ricinus communis) contains 90% ricinoleate, a hydroxy FA that is used in producing numerous industrial products. Castor diacylglycerol acyltransferase (RcDGAT) is a critical enzyme, as it catalyzes the terminal step in castor oil biosynthesis in which the products contain two or three ricinoleoyl moieties. We have isolated a cDNA encoding RcDGAT from developing castor seeds. Analysis of the sequence reveals that this cDNA encodes a protein of 521 amino acids with a molecular mass of 59.9 kDa. Although there are regions of high similarity to other plant DGAT coding sequences, there are sequences that distinguish it as well. Southern blot analysis suggests that the castor genome contains a single copy of RcDGAT. Analysis by reverse transcription-PCR reveals that the accumulation of the mRNA reaches its highest level at 19 d after pollination and declines thereafter. Expression of the full-length cDNA for RcDGAT in the yeast Saccharomyces cerevisiae strain INVSc1 results in sevenfold higher DGAT activity compared with controls. When different molecular species of DAG were provided as substrates to the microsomal mixture, the RcDGAT showed a greater preference to catalyze the transfer of oleate from [14C]oleoyl-CoA to diricinolein than to diolein and dipalmitolein. With the addition of 0.25 mM substrates, diricinolein gave 318 pmol/mg/min diricinoleoyloleoylglycerol (RRO), while diolein and dipalmitolein gave only about 195 pmol/mg/min of triolein (OOO) and 120 pmol/mg/min dipalmitoyleoylglycerol (PoPoO), respectively. This work will facilitate investigation of the role of RcDGAT in castor oil biosynthesis.

Non-aqueous reversed-phase high-performance liquid chromatography of synthetic triacylglycerols and diacylglycerols

Reversed-phase high-performance liquid chromatography (HPLC) methods were developed for the separation of molecular species of 45 synthetic triacylglycerols and diacylglycerols. These methods used linear gradients of methanol-isopropanol and UV detection at 205 nm as well as radioactive flow detection, which we add for metabolic studies. The elution orders of triacylglycerols and diacylglycerols depend on the polarity of their fatty acid constituents with elution time increasing as polarity decreases. The elution orders of triacylglycerols depending on their fatty acid constituents were as follows: ricinoliec acid<linolenic acid<palmitoleic acid<myristic acid<palmitelaidic acid<linoleic acid<linolelaidic acid<oleic acid<palmitic acid<elaidic acid<petroselinic acid<petroselaidic acid<stearic acid, while the elution orders of diacylglycerols were: palmitic acid<oleic acid<stearic acid. For both classes of glycerides, elution corresponded closely with chain length, degree of unsaturation and presence of polar groups and they were similar to the elution orders of fatty acids on an aqueous C18 HPLC which we reported recently: Other structural features also affect eltion order, as triacylglycerol containing a cis-fatty acid elutes slightly earlier than its isomer containing a trans-fatty acid. Additionally, higher polarity in the sn-2 position causes earlier elution; Diacylglycerol with a hydroxy group at sn-2 position of the glycerol backbone and triacylglycerol with a polar group on the fatty acid chain at sn-2 position elute slightly earlier than their respective sn-1(3) isomers.

Simple Methods to Detect Triacylglycerol Biosynthesis in a Yeast-Based Recombinant System

Standard methods to quantify the activity of triacylglycerol (TAG) synthesizing enzymes DGAT and PDAT (TAG-SE) require a sensitive but rather arduous laboratory assay based on radio-labeled substrates. Here we describe two straightforward methods to detect TAG production in baker’s yeast Saccharomyces cerevisiae. First we demonstrate that a quadruple knockout yeast strain deficient in storage lipids has a reduced growth rate in a medium supplemented with fatty acids. This phenotype is rescued by restoring TAG biosynthesis and can be thus used to select yeast cells expressing a recombinant TAG-SE. In the second method, the activity of the recombinant enzyme is measured in a fluorescent in situ assay using Nile red dye that is specific for neutral lipids. Correlation between Nile red fluorescence and enzyme activity is demonstrated with several mutants of a TAG synthesizing enzyme. This yeast live-cell-based assay is rapid, inexpensive, sensitive, and is amenable to high-throughput applications. The methods can be used for a variety of applications such as isolation of novel genes, directed evolution, gene-specific drug screening and will facilitate novel approaches in the research of TAG-SE.

A comparison of the conversion of 1-amino-2-ethylcyclopropane-1-carboxylic acid stereoisomers to 1-butene by pea epicotyls and by a cell-free system.

The characteristics of the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by pea (Pisum sativum L.) epicotyls and by pea epicotyl enzyme are compared. Of the four stereoisomers of 1-amino-2-ethylcyclopropane-1-carboxylic acid (AEC), only (1R,2S)-AEC is preferentially converted to 1-butene in pea epicotyls. This conversion is inhibited by ACC, indicating that butene production from (1R,2S)-AEC and ethylene production from ACC are catalyzed by the same enzyme. Furthermore, pea epicotyls efficiently convert ACC to ethylene with a low Km (66 μM) for ACC and do not convert 4-methylthio-2-oxo-butanoic acid (KMB) to ethylene, thus demonstrating high specificity for its substrate. In contrast, the reported pea epicotyl enzyme which catalyzes the conversion of ACC to ethylene had a high Km (389 mM) for ACC and readily converted KMB to ethylene. We show, moreover, that the pea enzyme catalyzes the conversion of AEC isomers to butene without stereodiscrimination. Because of its lack of stereospecificity, its low affinity for ACC and its utilization of KMB as a substrate, we conclude that the reported pea enzyme system is not related to the in-vivo ethylene-forming enzyme.

Gradient reversed-phase high-performance liquid chromatography of saturated, unsaturated and oxygenated free fatty acids and their methyl esters

Abstract Reversed-phase C18 high-performance liquid chromatography (HPLC) separation of 49 free fatty acids and their methyl esters is reported here. Separation is achieved using methanol/water gradients and detection by either UV absorbance or evaporative light scattering. The factors decreasing retention times are: keto group, hydroxy group, epoxy group, triple bond, shortened chain length (C2H4 shorter), cyclopropenyl group, double bond, and cyclopropanyl group, in approximate correspondence to the order of their decreasing polarity. Fatty acids with cis-double bond, cis-epoxy and threo-dihydroxy substituents elute slightly earlier than the corresponding trans-double bond, trans-epoxy and erythro-dihydroxy fatty acids. These geometric isomers are better separated as free fatty acids than their methyl esters. For monounsaturates, cis-double bond at C-10 of the methyl ester of octadecenoic acid decreased the retention times most, with slightly increasing retention time as the double bond shifts toward either end of the chain. The trans-double bond of methyl esters of octadecenoic acids showed similar results. The effect of single double bond position in the HPLC of free octadecenoic acids is similar except the shortest retention time occurs when the double bond is at C-11. Two of the C18 columns in series achieve the separation of double bond positional isomers (free fatty acids), cis-7-octadecenoic acid and petroselinic acid (cis-6) at a resolution Rs = 1.05.

Expression Profiles of Genes Involved in Fatty Acid and Triacylglycerol Synthesis in Castor Bean (Ricinus communis L.)

Castor seed triacylglycerols (TAGs) contain 90% ricinoleate (12-hydroxy-oleate) which has numerous industrial applications. Due to the presence of the toxin ricin and potent allergenic 2S albumins in the seed, it is desirable to produce ricinoleate from temperate oilseeds. To identify regulatory genes or genes for enzymes that may up-regulate multiple activities or entire pathways leading to the ricinoleate and TAG synthesis, we have analyzed expression profiles of 12 castor genes involved in fatty acid and TAG synthesis using quantitative reverse transcription-polymerase chain reaction technology. A collection of castor seeds with well-defined developmental stages and morphologies was used to determine the levels of mRNA, ricinoleate and TAG. The synthesis of ricinoleate and TAG occurred when seeds progressed to stages of cellular endosperm development. Concomitantly, most of the genes increased their expression levels, but showed various temporal expression patterns and different maximum inductions ranging from 4- to 43,000-fold. Clustering analysis of the expression data indicated five gene groups with distinct temporal patterns. We identified genes involved in fatty acid biosynthesis and transport that fell into two related clusters with moderate flat-rise or concave-rise patterns, and others that were highly expressed during seed development that displayed either linear-rise or bell-shaped patterns. Castor diacylglycerol acyltransferase 1 was the only gene having a higher expression level in leaf and a declining pattern during cellular endosperm development. The relationships among gene expression, cellular endosperm development and ricinoleate/TAG accumulation are discussed.

Regulation of diacylglycerol acyltransferase in developing seeds of castor.

We have previously reported the cloning of castor diacylglycerol acyltransferase (RcDGAT) based on its homology to other plant type 1 diacylglycerol acyltransferases (DGATs). To elucidate the physiological role of the RcDGAT, we have investigated the regulation of RcDGAT expression in developing seeds of castor. The RcDGAT transcript appeared at 12 d after pollination (DAP), reached the highest level at 26 DAP, and declined rapidly after that. However, the RcDGAT protein started to accumulate at 26 DAP, reached its peak at 47 DAP, then remained at this high level until 54 DAP. The significant difference between the expression of mRNA and protein indicates that gene expression of RcDGAT in maturing castor seeds is controlled at the posttranscriptional level. We found that DGAT activity measured in microsomal membranes isolated from seed at different stages of development was parallel to RcDGAT protein level, suggesting DGAT activity is mainly a function of the level of RcDGAT protein. We monitored the triacylglycerol (TG) composition and content during seed development. Compared with the overall rate of TG accumulation, DGAT activity appeared coincidently with the onset of lipid accumulation at 26 DAP; the highest DGAT activity occurred during the rapid phase of lipid accumulation at 40 DAP; and a decline in DGAT activity coincided with a decline in the accumulation rate of TG after 40 DAP. The ricinoleate-containing TG content was very low (only about 7%) in oil extracted from seeds before 19 DAP; however it increased up to about 77% of the oil at 26 DAP. The relative amount of triricinolein in oil at 26 DAP was 53 times higher than that at 19 DAP, and it was about 76% of the amount present in oil from mature castor seeds. The close correlation between profiles of RcDGAT activity and oil accumulation confirms the role of RcDGAT in castor oil biosynthesis.

Identification and Quantification of the Molecular Species of Acylglycerols in Castor Oil by HPLC Using ELSD

Abstract Sixteen molecular species of acylglycerols (AG) in castor oil have been identified and quantified. Evaporative light scattering detection (ELSD) responses of different amounts of the standards of molecular species of AG were nearly linear and similar. In general, the addition of a double bond and a hydroxyl group on the acyl chain and the shortening of the acyl chain decreased the ELSD response of AG slightly. The quantification of molecular species of AG was based on the percentage peak area in the HPLC chromatogram. Triricinolein (RRR) constituted about 71% of castor oil. The contents of the molecular species of diricinoleoyltriacylglycerol (RR‐TAG) were, in total, about 18% and were in the order of RRO (8.8%), RRL (6.6%), RRS (1.1%), RR‐lesqueroleate (0.67%), RRP (0.47%) and RRLn (0.15%). The level of R‐TAG was about 0.3%. The level of TAG containing no ricinoleate was less than 0.1%. Diricinoleoylglycerol, a diacylglycerol, was also identified and quantified (0.14%) in castor oil.

Development of a novel immuno-PCR assay for detection of ricin in ground beef, liquid chicken egg, and milk.

Reliable, sensitive, and high-throughput methods are essential for food defense, to detect foodborne contaminants and to facilitate remediation and recovery from potential toxin-related incidents. Ricin is a protein toxin that has been used for intentional contamination of foods in the past. In this study, we developed procedures for quantification of ricin in foods using immuno-PCR (IPCR). The direct adsorption of ricin onto the wells of a microtitration plate was compared with indirect immobilization via a capture antibody (sandwich IPCR). The latter procedure provided much greater sensitivity. We also compared a protocol with the immunoassay and PCR conducted in a single plate to a two-step procedure in which the PCR was conducted in a second plate, following release and transfer of the DNA marker. The two-step procedure proved 1,000-fold more sensitive for ricin detection, so this format was used to detect ricin in spiked samples of ground beef, chicken egg, and milk, and the results were compared with those obtained from enzyme-linked immunosorbent assay (ELISA). The IPCR had a limit of detection of 10 pg/ml in chicken egg and milk samples and 100 pg/ml in ground beef extracts. Comparable ELISA results were in the 1 to 10 ng/ml range. Thus, IPCR affords sensitivity that is 10-fold greater in the ground beef matrix, 100-fold greater in the milk, and 1,000-fold greater in the egg matrix than the sensitivity obtained by ELISA. Further optimization of the sandwich IPCR was performed by comparing various antibody combinations. Among the four formats investigated, the pAb-pAb combination yielded the lowest limit of detection (10 fg/ml).