Vernicia Fordii Research Articles

Hydrophobic‐Domain‐Dependent Protein–Protein Interactions Mediate the Localization of GPAT Enzymes to ER Subdomains

The endoplasmic reticulum (ER) is a dynamic organelle that consists of numerous regions or 'subdomains' that have discrete morphological features and functional properties. Although it is generally accepted that these subdomains differ in their protein and perhaps lipid compositions, a clear understanding of how they are assembled and maintained has not been well established. We previously demonstrated that two diacylglycerol acyltransferase enzymes (DGAT1 and DGAT2) from tung tree (Vernicia fordii) were located in different subdomains of ER, but the mechanisms responsible for protein targeting to these subdomains were not elucidated. Here we extend these studies by describing two glycerol-3-phosphate acyltransferase-like (GPAT) enzymes from tung tree, GPAT8 and GPAT9, that both colocalize with DGAT2 in the same ER subdomains. Measurement of protein-protein interactions using the split-ubiquitin assay revealed that GPAT8 interacts with itself, GPAT9 and DGAT2, but not with DGAT1. Furthermore, mutational analysis of GPAT8 revealed that the protein's first predicted hydrophobic region, which contains an amphipathic helix-like motif, is required for interaction with DGAT2 and for DGAT2-dependent colocalization in ER subdomains. Taken together, these results suggest that the regulation and organization of ER subdomains is mediated at least in part by higher-ordered, hydrophobic-domain-dependent homo- and hetero-oligomeric protein-protein interactions.

Biodiesel production from Tung oil and blended oil via ultrasonic transesterification process

The effects of ultrasonic irradiation time ( t US) on the transesterification yield ( Y F) of biodiesel from Tung ( Vernicia fordii) and blended oils with CH 3OH and KOH, and on some properties of biodiesel such as acid value (AV), iodine value (IV), kinematic viscosity (KV), density and cold filter plugging point (CFPP) were investigated. The blended oil is consisted of 20, 50 and 30% of Tung, canola and palm oils, respectively. The molar ratio of methanol to oil is 6:1 and the catalyst concentration is 1 wt.%. Temperature and ultrasonic frequency were kept at 20–30 °C and 25 kHz, respectively. The t US was set in the range of 1–30 min. The results showed that Y F reaches high value of 87–91% for Tung oil as t US ≥ 5 min, while of about 92–94% for blended oil as t US ≥ 1 min. At t US = 10 min, the properties of biodiesel produced from Tung oil are with AV of 0.11 mg KOH/g, IV of 159.36 mg I 2/100 g, KV of 9.17 mm 2/s, density of 905 kg/m 3 and CFPP of −16 °C, while those from blended oil are with AV of 0.11 mg KOH/g, IV of 120.35 mg I 2/100 g, KV of 5.54 mm 2/s, density of 887 kg/m 3 and CFPP of −5 °C. Comparing these values with the ASTM-D6751 standards with AV < 0.5 mg KOH/g, KV = 1.9–6 mm 2/s and density = 860–900 kg/m 3 pointed out that the Tung oil should be blended with other oils in order to produce biodiesel satisfying the biodiesel standards. Moreover, the results indicated that a certain enough time, say 5 min, is needed to provide sufficient cavity heating and mixing via ultrasonic wave ensuring good properties of biodiesel produced. The KV of biodiesel using blended oil decreases from 6.26 mm 2/s at t US = 1 min to 5.54 mm 2/s at t US = 5 min, thus meeting the ASTM-D6751 value of 1.9–6.0 mm 2/s. The information obtained in this study is useful for the proper use of Tung oil in conjunction with other edible oils for the production of biodiesel with satisfactory qualities and the rational design and operation of ultrasonically catalytic transesterification process.

Leaf lipids from Euphorbia lagascae Spreng. and Euphorbia lathyris L.

The Euphorbiaceae family is comprised of plant species that synthesise unusual fatty acids in their seed oils, e.g., Ricinus communis (ricinoleic or hydroxyoleic acid), Aleurites fordii (eleostearic acid) or Euphorbia lagascae (vernolic or epoxyoleic acid). Accumulation of triacylglycerols (TAGs) in plant leaves of many species can occur, although in smaller quantities than in seeds. This is the first report on leaf lipids in Euphorbia. Total lipid (TL) content ranged from 2274 to 5757 μg/g of fresh weight in Euphorbia lathyris and from 1562 to 9813 μg/g of fresh weight in E. lagascae. The first, fifth, tenth or fifteenth true leaves (each case from 2- to 40-day-old leaves) were sampled from plants grown in the open field and in the glasshouse at Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (Murcia, Spain). TAGs were separated by thin-layer chromatography (TLC) and represented up to 44.3% of TL in E. lagascae. The amount of TAG peaked in the first leaf and subsequently decreased with aging in all leaves. Microscope preparations revealed the presence of oil bodies (3–10 μm), possibly acting as intermediate storage products, in Euphorbia leaf mesophyll cells. Vernolic acid was identified in E. lagascae leaf lipids (0–25.7% of total fatty acids), but it was not incorporated into TAGs. These results imply that the active enzyme, diacylglycerol acyltransferase (DGAT), in the leaf of E. lagascae does not exhibit any selectivity towards the native unusual fatty acid.

Chemical Constituents from the Roots of &lt;I&gt;Vernicia fordii&lt;/I&gt;

Chemical Constituents from the Roots of &lt;I&gt;Vernicia fordii&lt;/I&gt;

F-BOX and oleosin: additional target genes for future metabolic engineering in tung trees?

Tung oil, produced from seeds of the tung tree ( Vernicia fordii), is a valuable industrial oil. Genetic engineering can improve the oil content, making it more suitable for use in biodiesel, and the pivotal role of DGAT2 in triacylglycerol synthesis has been extensively studied. Our data indicated, however, that DGAT2 expression decreases rapidly during the late period of oil accumulation, which raises the possibility that other key genes might play important roles in triacylglycerol synthesis. Construction of a tung kernel cDNA library and related biochemical analyses was carried out. Notably, F-BOX genes and oleosin-coding genes responsible for oil body structure were the most abundant ESTs in our cDNA library. All the results indicate that F-BOX and oleosin might be actively involved in tung lipid metabolism and serve as additional candidate genes for genetic engineering of the tung tree.

Identification and characterization of an acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) gene from the microalga O. tauri

In order to identify novel genes encoding enzymes involved in the terminal step of triacylglycerol (TAG) formation, a database search was carried out in the genome of the unicellular photoautotrophic green alga Ostreococcus tauri. The search led to the identification of three putative type 2 acyl-CoA:diacylglycerol acyltransferase-like sequences (DGAT; EC 2.3.1.20), and revealed the absence of any homolog to type 1 or type 3 DGAT sequence in the genome of O. tauri. For two of the cDNA sequences (OtDGAT2A and B) enzyme activity was detected by heterologous expression in Saccharomyces cerevisiae mutant strains with impaired TAG metabolism. However, activity of OtDGAT2A was too low for further analysis. Analysis of their amino acid sequences showed that they share limited identity with other DGAT2 from different plant species, such as Ricinus communis and Vernicia fordii with approximately 25 to 30% identity. Lipid analysis of the mutant yeast cells revealed that OtDGAT2B showed broad substrate specificity accepting saturated as well as mono- and poly-unsaturated acyl-CoAs as substrates.

Isolation and phytotoxicity of terpenes from Tectona grandis.

A study was carried out on the allelopathic potential of four forest species, Tectona grandis, Aleurites fordii, Gliricidia sepium, and Maytenus buxifolia. The most active species, T. grandis, was selected to perform a phytochemical study. A new compound, abeograndinoic acid, was isolated, and elucidation of its structure showed that this compound has an unusual carbon skeleton. A further 21 known terpenoids-including 4 sesquiterpenoids, 8 diterpenes and 9 triterpenes-also were isolated. A biosynthetic scheme for the presence of the new compound is proposed. Bioactivity profiles that used etiolated wheat coleoptiles and phytotoxicity bioassays on the isolated compounds were conducted. The compounds that presented the highest phytotoxic activity are the diterpenes 9 (2-oxokovalenic acid) and 12 (19-hydroxyferruginol).

Genetic diversity and association of ISSR markers with the eleostearic content in tung tree (Vernicia fordii)

Tung tree is a useful woody oil plant in the world. In this study, both the genetic diversity and biochemical traits were analyzed in order to improve the breeding methods on tung tree. The mean genetic similarity coefficient (Gs), the mean Nei’s gene diversity (h) and the mean Shannon’s information index (I) of tung tree were 0.7821, 0.2192 and 0.3424, respectively. In dendrogram based on UPGMA, 30 cultivars were divided into three groups, A, B and C, which displayed a descending trend on eleostearic content. A conclusion was drawn that ISSR makers associated with the biochemical trait, can be used in marker-associated breeding programs for tung crop improvement targeted at high eleostearic content variety.

The Tung Oil Boom in Australasia: a Network Perspective

Ideas about networks are explored in the context of the interest within the British Empire and the United States of America in planting Tung Oil trees (Aleurites fordii) during the 1920s and 1930s. Closer attention is paid to the Australian and New Zealand experience and short-lived enthusiasm for the search for seeds, the collation of information on growth rates, and the planting of Tung trees. The paper briefly distinguishes various types of network research in human geography and concludes by raising some questions about space and time in network approaches in the social sciences more generally.

Estimation of Soil Organic Carbon Stocks in Plantation Forest Soils of Northern Taiwan

The possibility of estimating the global carbon pools on the earth has attracted scientists for decades. In Taiwan, among remaining forested areas, 14% (about 420,000 ha) are plantation forests. There are few investigations of the soil organic carbon (SOC) pool of plantation forests in Taiwan. Nine plantation tree species, including 3 broadleaf species and 6 coniferous species were selected to estimate the SOC pools in selected plantation forest soils in northern Taiwan. The uncertainty of the estimates was examined, and strategies to obtain more-precise database for SOC pool management are proposed in this study. The results showed that only 2 Soil Orders, Inceptisols and Ultisols, were found at the study sites, and values of the soil bulk density (Bd) in broadleaf plantation forests were relatively higher than those in coniferous plantation forests. The stone contents were about 17 and 14% (0~30 cm) on average in the broadleaf and coniferous plantations, respectively. The average SOC was about 28 g kg^(-1) (0~30 cm) in the broadleaf plantations and about 48 g kg^(-1) (0~30 cm) in the coniferous plantations. The SOC pool was lowest in the Aleurites fordii plantation forest and highest in the Chamaecyparis obtusa plantation forest. We calculated that the average values of the SOC pool in the broadleaf plantation forest were about 6.5 (0~30 cm), 8.2 (0~50 cm), and 9.6 kg m^(-2) (0~100 cm). In coniferous plantation forests, they were about 7.4, 9.7, and 12 kg m^(-2), respectively. Estimation of the SOC pools in this study was potentially compromised by measurement errors of soil Bd, soil volume calculations, C determinations, and the stoniness of the soils. These sources of error might have introduced a bias, when comparing SOC contents among studies in the literature where different methodologies were applied. In conclusion, we suggest that the database of lower taxonomic categories (i.e., greater detail) for Soil Taxonomy and the SOC variability within and among pedons of the same soil type strongly need estimates of soil C stocks in Taiwan.

Key mediators modulating TAG synthesis and accumulation in woody oil plants

Woody oil plant is gaining increasing interest as substitute for petroleum-derived materials, and its enriched hydroxy and conjugated fatty acids for industrial applications. In pursuit of high-value and level oils, a better understanding of mechanisms regarding triacylglycerol (TAG) synthesis and accumulation is required. Here we first summarized the germplasm resources of woody oil plants, and the key mediators on TAG synthesis and accumulation, among which diacylglycerol acyltransferases (DGATs) is discussed for its clear role in TAG amount and composition. Furthermore TAG-accosiated proteins called oleosins are also discussed in depth due to their determination on the amount and size of oil bodies. Previously, two transcripts of oleosins were isolated from Vernicia fordii by us, and the homology of oleosins is analyzed. TAG accumulation is the result of multi-play action of the above mediators at varied levels. Furthermore, the channel of fatty acids flux also serves as a limiting factor for determination of the rate of TAG accumulation. The knowledge of key mediators modulating TAG accumulation will provide new insight on further metabolic engineering of oilseeds in woody plants.

Effects of forest plantations on rainfall redistribution and erosion in the red soil region of Southern China

AbstractTo evaluate the effects of different forest plantations on rainfall redistribution, we measured throughfall, stemflow, interception loss, surface runoff and soil loss from July 2004 to September 2005 in the three types of forest plantations Eucommia ulmoides, Vernicia fordii and Pinus massoniana. The results showed that differences in throughfall and stemflow between the three forest plantations were significant (p &lt; 0·05). Throughfall was highest in the V.fordii plantation and stemflow was highest in the E.ulmoides plantation. Throughfall plus stemflow below the E.ulmoides canopy was greater than that underneath the other forest types. Moreover, significant spatial variation in throughfall was observed. Throughfall in P.massoniana was 28·0–39·7% higher at a stem distance &lt; 60 cm or 11·5% lower at a stem distance &gt; 120 cm than in the other forests, but the difference was not significant between E.ulmoides and V.fordii. Moreover, the difference in throughfall at stem distances 60–120 cm was not significant between the different forest plantations. For E.ulmoides, throughfall under the peripheral crown part was 16·1% higher than that close to the stem. In contrast to E.ulmoides, P.massoniana had 26·8% lower throughfall under the peripheral crown part than close to the stem. No significant difference was found in throughfall for the various stem distances underneath V.fordii. Stemflow in E.ulmoides was 2–3 times higher than in the other forests (p &lt; 0·01). Interception loss accounted for 19·9% of gross rainfall for E.ulmoides, 20·8% for V.fordii and 27·2% for P.massoniana. Surface runoff and soil loss differed considerably among the three types of forest plantations. Annual runoff and total soil loss were lowest in the P.massoniana forest and highest in the V.fordii forest. This study indicated that P.massoniana, as a reforestation tree species, had the most positive effect on soil and water conservation among the three forest plantations. Copyright © 2008 John Wiley &amp; Sons, Ltd.

Genetic diversity and germplasm resource research on tung tree ( Vernicia fordii ) cultivars, investigated by inter-simple sequence repeats

Tung tree is an important woody oil-rich plant in the world. In order to determine the genetic diversity, germplasm resource and breeding method on tung tree, inter-simple sequence repeats (ISSR) was used to investigate the cultivars in China. Among the total 110 bands amplified with 12 primers, 90 were polymorphic. The mean genetic similarity coefficient (Gs), the mean Nei’s gene diversity (h), and the mean Shannon’s information index (I) of tung cultivars were 0.8273, 0.1770 and 0.2453, respectively. Both UPGMA cluster and PCA showed clear genetic relationship among the 64 tung cultivars. The multilocus marker system also yielded useful strategies for germplasm collection and conservation of tung.

The C-terminus of cytochrome b5 confers endoplasmic reticulum specificity by preventing spontaneous insertion into membranes

The molecular mechanisms that determine the correct subcellular localization of proteins targeted to membranes by tail-anchor sequences are poorly defined. Previously, we showed that two isoforms of the tung oil tree [Vernicia (Aleurites) fordii] tail-anchored Cb5 (cytochrome b5) target specifically to ER (endoplasmic reticulum) membranes both in vivo and in vitro [Hwang, Pelitire, Henderson, Andrews, Dyer and Mullen (2004) Plant Cell 16, 3002-3019]. In the present study, we examine the targeting of various tung Cb5 fusion proteins and truncation mutants to purified intracellular membranes in vitro in order to assess the importance of the charged CTS (C-terminal sequence) in targeting to specific membranes. Removal of the CTS from tung Cb5 proteins resulted in efficient binding to both ER and mitochondria. Results from organelle competition, liposome-binding and membrane proteolysis experiments demonstrated that removal of the CTS results in spontaneous insertion of tung Cb5 proteins into lipid bilayers. Our results indicate that the CTSs from plant Cb5 proteins provide ER specificity by preventing spontaneous insertion into incorrect subcellular membranes.

Tung Tree DGAT1 and DGAT2 Have Nonredundant Functions in Triacylglycerol Biosynthesis and Are Localized to Different Subdomains of the Endoplasmic Reticulum

Seeds of the tung tree (Vernicia fordii) produce large quantities of triacylglycerols (TAGs) containing approximately 80% eleostearic acid, an unusual conjugated fatty acid. We present a comparative analysis of the genetic, functional, and cellular properties of tung type 1 and type 2 diacylglycerol acyltransferases (DGAT1 and DGAT2), two unrelated enzymes that catalyze the committed step in TAG biosynthesis. We show that both enzymes are encoded by single genes and that DGAT1 is expressed at similar levels in various organs, whereas DGAT2 is strongly induced in developing seeds at the onset of oil biosynthesis. Expression of DGAT1 and DGAT2 in yeast produced different types and proportions of TAGs containing eleostearic acid, with DGAT2 possessing an enhanced propensity for the synthesis of trieleostearin, the main component of tung oil. Both DGAT1 and DGAT2 are located in distinct, dynamic regions of the endoplasmic reticulum (ER), and surprisingly, these regions do not overlap. Furthermore, although both DGAT1 and DGAT2 contain a similar C-terminal pentapeptide ER retrieval motif, this motif alone is not sufficient for their localization to specific regions of the ER. These data suggest that DGAT1 and DGAT2 have nonredundant functions in plants and that the production of storage oils, including those containing unusual fatty acids, occurs in distinct ER subdomains.

Conjugated fatty acids accumulate to high levels in phospholipids of metabolically engineered soybean and Arabidopsis seeds

Expression of Δ 12-oleic acid desaturase-related fatty acid conjugases from Calendula officinalis, Momordica charantia, and Vernicia fordii in seeds of soybean ( Glycine max) or an Arabidopsis thaliana fad3/fae1 mutant was accompanied by the accumulation of the conjugated fatty acids calendic acid or α-eleostearic acid to amounts as high as 20% of the total fatty acids. Conjugated fatty acids, which are synthesized from phosphatidylcholine (PC)-linked substrates, accumulated in PC and phosphatidylethanolamine, and relative amounts of these fatty acids were higher in PC than in triacylglycerol (TAG) in the transgenic seeds. The highest relative amounts of conjugated fatty acids were detected in PC from seeds of soybean and A. thaliana that expressed the C. officinalis and M. charantia conjugases, where they accounted for nearly 25% of the fatty acids of this lipid class. In these seeds, >85% of the conjugated fatty acids in PC were detected in the sn-2 position, and these fatty acids were also enriched in the sn-2 position of TAG. In marked contrast to the transgenic seeds, conjugated fatty acids composed <1.5% of the fatty acids in PC from seeds of five unrelated species that naturally synthesize a variety of conjugated fatty acid isomers, including seeds that accumulate conjugated fatty acids to >80% of the total fatty acids. These results suggest that soybean and A. thaliana seeds are deficient in their metabolic capacity to selectively catalyze the flux of conjugated fatty acids from their site of synthesis on PC to storage in TAG.

Lack of chemopreventive effects of α-eleostearic acid on 7,12-dimethylbenz[ a]anthracene (DMBA) and 1,2-dimethylhydrazine (DMH)-induced mammary and colon carcinogenesis in female Sprague–Dawley rats

α-Eleostearic acid is one of the conjugated linolenic acids from tung oil, which is obtained from the seeds of Aleurites fordii. The effects of dietary α-eleostearic acid (18:3, n-5) on the post-initiation period of 7,12-dimethylbenz[ a]anthracene (DMBA) and 1,2-dimethylhydrazine (DMH)-induced mammary and colon carcinogenesis were examined using female Sprague–Dawley (SD) rats. For initiation, rats were given subcutaneous injections of 40 mg/kg body weight (5 times) and 20 mg/kg body weight (3 times) of DMH during the age of 6–8 weeks and a single intragastric administration of 50 mg/kg body weight of DMBA at 9 weeks. Then, the animals were treated with 0%, 0.01%, 0.1% or 1.0% α-eleostearic acid for 34 weeks. Control rats received the basal diet alone or 1.0% α-eleostearic acid without prior initiation treatment. All surviving animals were killed at week 37 of the experiment. There were no statistically significant alterations in any of the parameters for either mammary or colon tumors. These results thus indicate that α-eleostearic acid does not exert clear modification effects on DMBA and DMH-induced mammary and colon carcinogenesis, at least under the present experimental conditions.

Cloning, functional analysis, and subcellular localization of two isoforms of NADH:cytochrome b5 reductase from developing seeds of tung ( Vernicia fordii)

Two genes and the corresponding cDNAs for NADH:cytochrome b 5 reductase ( VfCBR1A and VfCBR1B) from tung ( Vernicia fordii) were cloned and characterized. Both tung genes are expressed at similar levels in various organs throughout the plant, but differ substantially in their genomic architecture. Phylogenetic comparisons of many cloned and putative CBR genes from plants and yeast revealed two general classes of sequences. The separation of the classes likely reflects differences in the subcellular targeting of the two types of proteins. Immunofluorescence analyses of tobacco BY-2 cells containing transiently expressed tung CBR1A, CBR1B, or Arabidopsis thaliana CBR revealed definitive targeting of the proteins to the endoplasmic reticulum while a previously uncharacterized Arabidopsis CBR protein was targeted specifically to mitochondria. After overexpression in Saccharomyces cerevisiae, VfCBR1A was enzymatically active, and like its Arabidopsis ortholog, displayed strict specificity for NADH as the reductant. The subcellular localization and biochemical properties of the tung enzymes are consistent with a potential role in fatty acid desaturation and conjugation.

Sensitive assay for hormone-sensitive lipase using NBD-labeled monoacylglycerol to detect low activities in rat adipocytes

The recent finding that p-nitrobenzofurazan (NBD)-FA is incorporated into and released from the acylglycerols of isolated rat adipocytes in an insulin-sensitive manner [G. Muller, H. Jordan, C. Jung, H. Kleine, and S. Petry. 2003. Biochimie. 85: 1245-1246] suggests that NBD-FA-labeled acylglycerols are cleaved by rat adipocyte hormone-sensitive lipase (HSL) in vivo. In the present study, we developed a continuous, sensitive in vitro lipase assay using a monoacylglycerol (MAG) containing NBD (NBD-MAG). NBD-MAG was found to provide an efficient substrate for rat adipocyte and human recombinant HSL. Ultrasonic treatment applied in the presence of phospholipids leads to the incorporation of NBD-MAG into the phospholipid liposomes and to a concomitant change of its spectrophotometric properties. The enzymatic release of NBD-FA and its dissociation from the carrier liposomes is accompanied by the recovery of the original spectrophotometric characteristics. The rate of lipolysis was monitored by measuring the increase in optical density at 481 nm, which was found to be linear with time and linearly proportional to the amount of lipase added. To assess the specific activity of recombinant HSL, we determined the molar extinction coefficient of NBD-FA under the assay conditions. This convenient assay procedure based on NBD-MAG should facilitate the search for small molecule HSL inhibitors.

Novel targeting signals mediate the sorting of different isoforms of the tail-anchored membrane protein cytochrome b5 to either endoplasmic reticulum or mitochondria.

Tail-anchored membrane proteins are a class of proteins that are targeted posttranslationally to various organelles and integrated by a single segment of hydrophobic amino acids located near the C terminus. Although the localization of tail-anchored proteins in specific subcellular compartments in plant cells is essential for their biological function, the molecular targeting signals responsible for sorting these proteins are not well defined. Here, we describe the biogenesis of four closely related tung (Aleurites fordii) cytochrome b5 isoforms (Cb5-A, -B, -C, and -D), which are small tail-anchored proteins that play an essential role in many cellular processes, including lipid biosynthesis. Using a combination of in vivo and in vitro assays, we show that Cb5-A, -B, and -C are targeted exclusively to the endoplasmic reticulum (ER), whereas Cb5-D is targeted specifically to mitochondrial outer membranes. Comprehensive mutational analyses of ER and mitochondrial Cb5s revealed that their C termini, including transmembrane domains (TMD) and tail regions, contained several unique physicochemical and sequence-specific characteristics that defined organelle-specific targeting motifs. Mitochondrial targeting of Cb5 was mediated by a combination of hydrophilic amino acids along one face of the TMD, an enrichment of branched beta-carbon-containing residues in the medial portion of the TMD, and a dibasic -R-R/K/H-x motif in the C-terminal tail. By contrast, ER targeting of Cb5 depended primarily upon the overall length and hydrophobicity of the TMD, although an -R/H-x-Y/F- motif in the tail was also a targeting determinant. Collectively, the results presented provide significant insight into the early biogenetic events required for entry of tail-anchored proteins into either the ER or mitochondrial targeting pathways.