Genome-Wide Identification of B-Box Gene Family and Candidate Light-Related Member Analysis of Tung Tree (Vernicia fordii).

Light is one of the most important environmental factors for plant growth. In the production process of tung oil tree cultivation, due to the inappropriate growth of shading conditions, the lower branches are often dry and dead, which seriously affects the yield of tung oil trees. However, little is known about the key factors of light-induced tree photomorphogenesis. In this study, a total of 22 VfBBX family members were identified to provide a reference for candidate genes in tung tree seedlings. All members of the VfBBX family have different numbers of highly conserved B-box domains or CCT domains. Phylogenetic evolution clustered the VfBBX genes into four categories, and the highest density of members was on chromosome 6. Interspecific collinearity analysis suggested that there were six pairs of duplicate genes in VfBBX members, but the expression levels of all family members in different growth and development stages of the tung tree were significantly divergent. After different degrees of shading treatment and physiological data determination of tung tree seedlings, the differential expression level and chlorophyll synthesis genes correlation analysis revealed that VfBBX9 was a typical candidate nuclear localization transcription factor that was significantly differentially expressed in light response. This study systematically identified the VfBBX gene family and provided a reference for studying its molecular function, enhanced the theoretical basis for tung tree breeding, and identified excellent varieties.

Genome-wide identification and transcriptional profiling of the basic helix-loop-helix gene family in tung tree (Vernicia fordii).

The basic helix-loop-helix (bHLH) transcription factor gene family is one of the largest gene families and is extensively involved in plant growth, development, biotic and abiotic stress responses. Tung tree (Vernicia fordii) is an economically important woody oil plant that produces tung oil rich in eleostearic acid. However, the characteristics of the bHLH gene family in the tung tree genome are still unclear. Hence, VfbHLHs were first searched at a genome-wide level, and their expression levels in various tissues or under low temperature were investigated systematically. In this study, we identified 104 VfbHLHs in the tung tree genome, and these genes were classified into 18 subfamilies according to bHLH domains. Ninety-eight VfbHLHs were mapped to but not evenly distributed on 11 pseudochromosomes. The domain sequences among VfbHLHs were highly conserved, and their conserved residues were also identified. To explore their expression, we performed gene expression profiling using RNA-Seq and RT-qPCR. We identified five, 18 and 28 VfbHLH genes in female flowers, male flowers and seeds, respectively. Furthermore, we found that eight genes (VfbHLH29, VfbHLH31, VfbHLH47, VfbHLH51, VfbHLH57, VfbHLH59, VfbHLH70, VfbHLH72) were significant differential expressed in roots, leaves and petioles under low temperature stress. This study lays the foundation for future studies on bHLH gene cloning, transgenes, and biological mechanisms.

Evolution and function of ubiquitin-specific proteases (UBPs): Insight into seed development roles in plants.

The tung oil produced by the tung tree (Vernicia fordii) provides resources for the manufacture of biodiesel. Ubiquitin-specific proteases (UBPs) are the largest group of deubiquitinases and play key roles in regulating development and stress responses. Here, 21 UBPs were identified in V. fordii, roughly one-half the number found in Manihot esculenta and Hevea brasiliensis. Most UBP duplications are produced from whole-genome duplication (WGD), and significant differences in gene retention existed among Euphorbiaceae. The great majority of UBP-containing blocks in V. fordii, V. montana, Ricinus communis, and Jatropha curcas exhibited extensive conservation with the duplicated regions of M. esculenta and H. brasiliensis. These blocks formed 14 orthologous groups, indicating they shared WGD with UBPs in M. esculenta and H. brasiliensis, but most of these UBPs copies were lost. The UBP orthologs contained significant functional divergence which explained the susceptibility of V. fordii to Fusarium wilt and the resistance of V. montana to Fusarium wilt. The expression patterns and experiments suggested that Vf03G1417 could affect the seed-related traits and positively regulate the seed oil accumulation. This study provided important insights into the evolution of UBPs in Euphorbiaceae and identified important candidate VfUBPs for marker-assisted breeding in V. fordii.

Dissection of leucine-rich repeat receptor-like protein kinases: insight into resistance to Fusarium wilt in tung tree.

The tung tree is a woody oil plant native to China and widely distributed in the subtropics. The three main species commonly known as Vernicia are V. fordii, V. montana, and V. cordata. The growth and development of V. fordii are affected by a large number of plant pathogens, such as Fusarium wilt caused by Fusarium sp. In contrast, V. montana shows significant resistance to Fusarium wilt. The leucine-rich repeat receptor-like protein kinase (LRR-RLK) is the largest class of receptor-like kinases associated with plant resistance to Fusarium wilt. Here, we identified 239 VmLRR-RLKs in V. montana, and found that there were characteristic domains of resistance to Fusarium wilt in them. Phylogenetic analysis suggested that the VmLRR-RLKs are divided into 14 subfamilies, indicating that homologous genes in the same group may have similar functions. Chromosomal localization analysis showed that VmLRR-RLKs were unevenly distributed on chromosomes, and segment duplications were the main reason for the expansion of VmLRR-RLK family members. The transcriptome data showed that six orthologous pairs were up-regulated in V. montana in response to Fusarium wilt, while the corresponding orthologous genes showed low or no expression in V. fordii in resistance Fusarium wilt, further indicating the important role of LRR-RLKs in V. montana's resistance to infection by Fusarium spp. Our study provides important reference genes for the future use of molecular breeding to improve oil yield and control of Fusarium wilt in tung tree.

Deciphering the roles of leucine-rich repeat receptor-like protein kinases (LRR-RLKs) in response to Fusarium wilt in the Vernicia fordii (Tung tree).

Leucine-rich repeat receptor-like protein kinases (LRR-RLKs) are vital for plant growth and development, signal transduction, immunity, and play diverse roles in plant defense responses. However, the LRR-RLK genes have not been systematically studied in Vernicia fordii (tung tree), especially its response to Fusarium wilt. Here, we carried out an integrative analysis of LRR-RLKs among five Euphorbiaceae species: Hevea brasiliensis (rubber tree), Manihot esculenta (cassava), Jatropha curcas (physic nut), Ricinus communis (castor bean), and V. fordii, which contained 223, 311, 186, 138, and 167 LRR-RLKs, respectively. Maximum-likelihood tree was estimated using LRR-RLKs of Arabidopsis thaliana as a template, and they allowed us to divide Euphorbiaceae LRR-RLKs into 22 groups. There are 126 segmental and 30 tandem duplications in these Euphorbiaceae genomes by synteny analysis. The tissue-specific expression patterns revealed that V. fordii LRR-RLKs (VfLRR-RLKs) were differentially expressed in various tissues, and some of them exhibited specific expression in meristems tissues, which suggested their potential functions during organ formation and cell fate specification. Two VfLRR-RLK pairs (Vf01G2125 and Vf03G1740, Vf06G2687 and Vf10G1659), which generated by tandem duplication events, were associated with possible resistance to Fusarium wilt infection. The qRT-PCR confirmed these four VfLRR-RLKs contained opposite expression profiles during pathogen infection in V. fordii and V. montana. Taken together, our data systematically analyzed the LRR-RLK family in Euphorbiaceae genomes for the first time. We highlight the putative roles of VfLRR-RLKs in response to Fusarium wilt infection, and VfLRR-RLKs may be further applied in marker-assisted breeding to control Fusarium wilt in V. fordii.

Hidden in plain sight: Systematic investigation of Leucine-rich repeat containing genes unveil the their regulatory network in response to Fusarium wilt in tung tree.

Leucine-rich repeat-containing genes (LRRs) have been reported to play important roles in responses to diseases. However, we poorly understood the response of LRRs to Fusarium wilt infection in tung tree (Vernicia fordii), which is an important economic tree. Here, 437 LRR-containing proteins containing nine types of LRR domains were identified in V. fordii genome. Phylogenetic analysis suggested that nine types of LRR domains could not be divided into separate classes, implying that these LRR domains had a common origin. Totally, 27 LRRs were related to possible resistance to Fusarium wilt after 2, 8, and 13 days post-inoculation. We further found that Vf06G1605 was up-regulate under Fusarium wilt infection after these three time points, Vf10G1602 and Vf02G1413 were up-regulated at 8, and 13 dpi, while Vf07G2320 was down-regulated at these three time points. The WGCNA and promoter elements suggested that WRKY possibly regulate the responses of LRRs to Fusarium wilt infection. This study highlighted the phylogeny and function of LRRs in V. fordii and provided a systematic analysis of these genes in the V. fordii genome. Our results presented here might clearly illustrate physiological mechanisms of resistance to Fusarium wilt infection and the target of marker-assisted breeding in V. fordii.

Tung Tree (Vernicia fordii) Genome Provides A Resource for Understanding Genome Evolution and Improved Oil Production.

Tung tree (Vernicia fordii) is an economically important woody oil plant that produces tung oil rich in eleostearic acid. Here, we report a high-quality chromosome-scale genome sequence of tung tree. The genome sequence was assembled by combining Illumina short reads, Pacific Biosciences single-molecule real-time long reads, and Hi-C sequencing data. The size of tung tree genome is 1.12 Gb, with 28,422 predicted genes and over 73% repeat sequences. The V. fordii underwent an ancient genome triplication event shared by core eudicots but no further whole-genome duplication in the subsequent ca. 34.55 million years of evolutionary history of the tung tree lineage. Insertion time analysis revealed that repeat-driven genome expansion might have arisen as a result of long-standing long terminal repeat retrotransposon bursts and lack of efficient DNA deletion mechanisms. The genome harbors 88 resistance genes encoding nucleotide-binding sites; 17 of these genes may be involved in early-infection stage of Fusarium wilt resistance. Further, 651 oil-related genes were identified, 88 of which are predicted to be directly involved in tung oil biosynthesis. Relatively few phosphoenolpyruvate carboxykinase genes, and synergistic effects between transcription factors and oil biosynthesis-related genes might contribute to the high oil content of tung seed. The tung tree genome constitutes a valuable resource for understanding genome evolution, as well as for molecular breeding and genetic improvements for oil production.

Molecular mechanism of the extended oil accumulation phase contributing to the high seed oil content for the genotype of tung tree (Vernicia fordii).

BACKGROUND: Oil from seeds of the tung tree (Vernicia fordii) has unique drying properties that are industrially important. We found that the extended oil accumulation period was related to the high seed oil content at maturity among tung tree population. In order to understand the molecular mechanism underlying the high oil content in tung tree seed, Tree H and L were adopted for the further investigation, with seed oil content of about 70 and 45%, respectively. We compared the transcriptomic changes of seed at various times during oil accumulation between the two trees. RESULTS: Transcriptomes analysis revealed that many genes involved in glycolysis, fatty acid synthesis, and tri-acyl glyceride assembly still kept high expression in the late period of seed oil accumulation for Tree H only. Many genes in fatty acid degradation pathway were largely up regulated in the late period of seed oil accumulation for Tree L only. Four transcription factors related to fatty acid biosynthesis had different expression pattern in the seed oil accumulation period for the two trees. WRI1 was down regulated and kept the low expression in the late period of seed oil accumulation for the two trees. PII, LEC1 and LEC1-LIKE extended the high expression in the late period of seed oil accumulation in Tree H only. CONCLUSIONS: The continued accumulation of oil in the late period of seed oil accumulation for Tree H was associated with relatively high expression of the relevant genes in glycolysis, fatty acid synthesis and tri-acyl glyceride assembly. PII, LEC1, and LEC1-LIKE rather than WRI1 should play an important role in the oil continual accumulation in the late period of seed oil accumulation in Tree H. This study provides novel insight into the variation in seed oil content and informs plant breeding strategies to maximize oil yield.

The complete chloroplast genome sequence of tung tree (Vernicia fordii): Organization and phylogenetic relationships with other angiosperms.

Tung tree (Vernicia fordii) is an economically important tree widely cultivated for industrial oil production in China. To better understand the molecular basis of tung tree chloroplasts, we sequenced and characterized its genome using PacBio RS II sequencing platforms. The chloroplast genome was sequenced with 161,528 bp in length, composed with one pair of inverted repeats (IRs) of 26,819 bp, which were separated by one small single copy (SSC; 18,758 bp) and one large single copy (LSC; 89,132 bp). The genome contains 114 genes, coding for 81 protein, four ribosomal RNAs and 29 transfer RNAs. An expansion with integration of an additional rps19 gene in the IR regions was identified. Compared to the chloroplast genome of Jatropha curcas, a species from the same family, the tung tree chloroplast genome is distinct with 85 single nucleotide polymorphisms (SNPs) and 82 indels. Phylogenetic analysis suggests that V. fordii is a sister species with J. curcas within the Eurosids I. The nucleotide sequence provides vital molecular information for understanding the biology of this important oil tree.

Molecular cloning and expression analysis of a novel BCCP subunit gene from Aleurites moluccana.

Aleurites moluccana L. is grown as a roadside tree in southern China and the oil content of its seed is higher than other oil plants, such as Jatropha curcas and Camellia oleifera. A. moluccana is considered a promising energy plant because its seed oil could be used to produce biodiesel and bio-jet fuel. In addition, the bark, leaves, and kernels of A. moluccana have various medical and commercial uses. Here, a novel gene coding the biotin carboxyl carrier protein subunit (BCCP) was cloned from A. moluccana L. using the homology cloning method combined with rapid amplification of cDNA end (RACE) technology. The isolated full-length cDNA sequence (designated AM-accB) was 1188 bp, containing a 795-bp open reading frame coding for 265 amino acids. The deduced amino acid sequence of AM-accB contained a biotinylated domain located between amino acids 190 and 263. A. moluccana BCCP shows high identity at the amino acid level to its homologues in other higher plants, such as Vernicia fordii, J. curcas, and Ricinus communis (86, 77, and 70%, respectively), which all contain conserved domains for ACCase activity. The expression of the AM-accB gene during the middle stage of development and maturation in A. moluccana seeds was higher than that in early and later stages. The expression pattern of the AM-accB gene is very similar to that of the oil accumulation rate.

Effects of tung oilseed FAD2 and DGAT2 genes on unsaturated fatty acid accumulation in Rhodotorula glutinis and Arabidopsis thaliana.

Genetic engineering to produce valuable lipids containing unsaturated fatty acids (UFAs) holds great promise for food and industrial applications. Efforts to genetically modify plants to produce desirable UFAs with single enzymes, however, have had modest success. The key enzymes fatty acid desaturase (FAD) and diacylglycerol acyltransferase (DGAT) are responsible for UFA biosynthesis (a push process) and assembling fatty acids into lipids (a pull process) in plants, respectively. To examine their roles in UFA accumulation, VfFAD2 and VfDGAT2 genes cloned from Vernicia fordii (tung tree) oilseeds were conjugated and transformed into Rhodotorula glutinis and Arabidopsis thaliana via Agrobacterium tumefaciens. Real-time quantitative PCR revealed variable gene expression levels in the transformants, with a much higher level of VfDGAT2 than VfFAD2. The relationship between VfFAD2 expression and linoleic acid (C18:2) increases in R. glutinis (R (2) = 0.98) and A. thaliana (R (2) = 0.857) transformants was statistically linear. The VfDGAT2 expression level was statistically correlated with increased total fatty acid content in R. glutinis (R (2) = 0.962) and A. thaliana (R (2) = 0.8157) transformants. With a similar expression level between single- and two-gene transformants, VfFAD2-VfDGAT2 co-transformants showed a higher linolenic acid (C18:3) yield in R. glutinis (174.36 % increase) and A. thaliana (14.61 % increase), and eicosatrienoic acid (C20:3) was enriched (17.10 % increase) in A. thaliana. Our data suggest that VfFAD2-VfDGAT2 had a synergistic effect on UFA metabolism in R. glutinis, and to a lesser extent, A. thaliana. These results show promise for further genetic engineering of plant lipids to produce desirable UFAs.

Fatty acid profile and unigene-derived simple sequence repeat markers in tung tree (Vernicia fordii).

Tung tree (Vernicia fordii) provides the sole source of tung oil widely used in industry. Lack of fatty acid composition and molecular markers hinders biochemical, genetic and breeding research. The objectives of this study were to determine fatty acid profiles and develop unigene-derived simple sequence repeat (SSR) markers in tung tree. Fatty acid profiles of 41 accessions showed that the ratio of α-eleostearic acid was increasing continuously with a parallel trend to the amount of tung oil accumulation while the ratios of other fatty acids were decreasing in different stages of the seeds and that α-eleostearic acid (18∶3) consisted of 77% of the total fatty acids in tung oil. Transcriptome sequencing identified 81,805 unigenes from tung cDNA library constructed using seed mRNA and discovered 6,366 SSRs in 5,404 unigenes. The di- and tri-nucleotide microsatellites accounted for 92% of the SSRs with AG/CT and AAG/CTT being the most abundant SSR motifs. Fifteen polymorphic genic-SSR markers were developed from 98 unigene loci tested in 41 cultivated tung accessions by agarose gel and capillary electrophoresis. Genbank database search identified 10 of them putatively coding for functional proteins. Quantitative PCR demonstrated that all 15 polymorphic SSR-associated unigenes were expressed in tung seeds and some of them were highly correlated with oil composition in the seeds. Dendrogram revealed that most of the 41 accessions were clustered according to the geographic region. These new polymorphic genic-SSR markers will facilitate future studies on genetic diversity, molecular fingerprinting, comparative genomics and genetic mapping in tung tree. The lipid profiles in the seeds of 41 tung accessions will be valuable for biochemical and breeding studies.

Identification, classification and differential expression of oleosin genes in tung tree (Vernicia fordii).

Triacylglycerols (TAG) are the major molecules of energy storage in eukaryotes. TAG are packed in subcellular structures called oil bodies or lipid droplets. Oleosins (OLE) are the major proteins in plant oil bodies. Multiple isoforms of OLE are present in plants such as tung tree (Vernicia fordii), whose seeds are rich in novel TAG with a wide range of industrial applications. The objectives of this study were to identify OLE genes, classify OLE proteins and analyze OLE gene expression in tung trees. We identified five tung tree OLE genes coding for small hydrophobic proteins. Genome-wide phylogenetic analysis and multiple sequence alignment demonstrated that the five tung OLE genes represented the five OLE subfamilies and all contained the "proline knot" motif (PX5SPX3P) shared among 65 OLE from 19 tree species, including the sequenced genomes of Prunus persica (peach), Populus trichocarpa (poplar), Ricinus communis (castor bean), Theobroma cacao (cacao) and Vitis vinifera (grapevine). Tung OLE1, OLE2 and OLE3 belong to the S type and OLE4 and OLE5 belong to the SM type of Arabidopsis OLE. TaqMan and SYBR Green qPCR methods were used to study the differential expression of OLE genes in tung tree tissues. Expression results demonstrated that 1) All five OLE genes were expressed in developing tung seeds, leaves and flowers; 2) OLE mRNA levels were much higher in seeds than leaves or flowers; 3) OLE1, OLE2 and OLE3 genes were expressed in tung seeds at much higher levels than OLE4 and OLE5 genes; 4) OLE mRNA levels rapidly increased during seed development; and 5) OLE gene expression was well-coordinated with tung oil accumulation in the seeds. These results suggest that tung OLE genes 1-3 probably play major roles in tung oil accumulation and/or oil body development. Therefore, they might be preferred targets for tung oil engineering in transgenic plants.

Developmental regulation of diacylglycerol acyltransferase family gene expression in tung tree tissues.

Diacylglycerol acyltransferases (DGAT) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGAT genes have been identified in numerous organisms. Multiple isoforms of DGAT are present in eukaryotes. We previously cloned DGAT1 and DGAT2 genes of tung tree (Vernicia fordii), whose novel seed TAGs are useful in a wide range of industrial applications. The objective of this study was to understand the developmental regulation of DGAT family gene expression in tung tree. To this end, we first cloned a tung tree gene encoding DGAT3, a putatively soluble form of DGAT that possesses 11 completely conserved amino acid residues shared among 27 DGAT3s from 19 plant species. Unlike DGAT1 and DGAT2 subfamilies, DGAT3 is absent from animals. We then used TaqMan and SYBR Green quantitative real-time PCR, along with northern and western blotting, to study the expression patterns of the three DGAT genes in tung tree tissues. Expression results demonstrate that 1) all three isoforms of DGAT genes are expressed in developing seeds, leaves and flowers; 2) DGAT2 is the major DGAT mRNA in tung seeds, whose expression profile is well-coordinated with the oil profile in developing tung seeds; and 3) DGAT3 is the major form of DGAT mRNA in tung leaves, flowers and immature seeds prior to active tung oil biosynthesis. These results suggest that DGAT2 is probably the major TAG biosynthetic isoform in tung seeds and that DGAT3 gene likely plays a significant role in TAG metabolism in other tissues. Therefore, DGAT2 should be a primary target for tung oil engineering in transgenic organisms.

Molecular properties of the class III subfamily of acyl-coenyzme A binding proteins from tung tree (Vernicia fordii).

Acyl-CoA binding proteins (ACBPs) have been identified in most branches of life, and play various roles in lipid metabolism, among other functions. Plants contain multiple classes of ACBP genes. The most diverse group is the class III proteins. Tung tree (Vernicia fordii) contains two such genes, designated VfACBP3A and VfACBP3B. The two proteins are significantly different in length and sequence. Analysis of tung ACBP3 genes revealed significant evolution, suggesting relatively ancient divergence of the two genes from a common ancestor. Phylogenetic comparisons of multiple plant class III proteins suggest that this group is the most evolutionarily dynamic class of ACBP. Both tung ACBP3 genes are expressed at similar levels in most tissues tested, but ACBP3A is stronger in leaves. Three-dimensional modeling predictions confirmed the presence of the conserved four α-helix bundle acyl-CoA binding (ACB); however, other regions of these proteins likely fold much differently. Acyl-CoA binding assays revealed different affinities for different acyl-CoAs, possibly contradicting the redundancy of function suggested by the gene expression studies. Subcellular targeting of transiently-expressed plant ACBP3 proteins contradicted earlier studies, and suggested that at least some class III ACBPs may be predominantly targeted to endoplasmic reticulum membranes, with little or no targeting to the apoplast.

Selection of reliable reference genes for gene expression studies using real-time PCR in tung tree during seed development.

Quantitative real-time PCR (RT-qPCR) has become an accurate and widely used technique to analyze expression levels of selected genes. It is very necessary to select appropriate reference genes for gene expression normalization. In the present study, we assessed the expression stability of 11 reference genes including eight traditional housekeeping genes and three novel genes in different tissues/organs and developing seeds from four cultivars of tung tree. All 11 reference genes showed a wide range of Ct values in all samples, indicating that they differently expressed. Three softwares--geNorm, NormFinder and BestKeeper--were used to determine the stability of these references except for ALB (2S albumin), which presented a little divergence. The results from the three softwares showed that ACT7 (Actin7a), UBQ (Ubiquitin), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and EF1α (elongation factor 1-α) were the most stable reference genes across all of the tested tung samples and tung developing seeds, while ALB (2S albumin) was unsuitable as internal controls. ACT7, EF1ß (elongation factor1-beta), GAPDH and TEF1 (transcription elongation factor 1) were the top four choices for different tissues/organs whereas LCR69 did not favor normalization of RT-qPCR in these tissues/organs. Meanwhile, the expression profiles of FAD2 and FADX were realized using stable reference genes. The relative quantification of the FAD2 and FADX genes varied according to the internal controls and the number of internal controls. The results further proved the importance of the choice of reference genes in the tung tree. These stable reference genes will be employed in normalization and quantification of transcript levels in future expression studies of tung genes.

Expression and purification of recombinant tung tree diacylglycerol acyltransferase 2.

Diacylglycerol acyltransferases (DGATs) esterify sn-1,2-diacylglycerol with a long-chain fatty acyl-CoA, the last and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. At least 74 DGAT2 sequences from 61 organisms have been identified, but the expression of any DGAT2 as a partial or full-length protein in Escherichia coli had not been reported. The main objective of this study was to express and purify recombinant DGAT2 (rDGAT2) from E. coli for antigen production with a minor objective to compare rDGAT2 expression in yeast. A plasmid was engineered to express tung tree DGAT2 fused to maltose binding protein and poly-histidine (His) affinity tags. Immunoblotting showed that rDGAT2 was detected in the soluble, insoluble, and membrane fractions. The rDGAT2 in the soluble fraction was partially purified by amylose resin, nickel-nitrilotriacetic agarose (Ni-NTA) beads, and tandem affinity chromatography. Multiple proteins co-purified with rDGAT2. Size exclusion chromatography estimated the size of the rDGAT2-enriched fraction to be approximately eight times the monomer size. Affinity-purified rDGAT2 fractions had a yellow tint and contained fatty acids. The rDGAT2 in the insoluble fraction was partially solubilized by seven detergents with SDS being the most effective. Recombinant DGAT2 was purified to near homogeneity by SDS solubilization and Ni-NTA affinity chromatography. Mass spectrometry identified rDGAT2 as a component in the bands corresponding to the monomer and dimer forms as observed by SDS-PAGE. Protein bands with monomer and dimer sizes were also observed in the microsomal membranes of Saccharomyces cerevisiae expressing hemagglutinin-tagged DGAT2. Nonradioactive assay showed TAG synthesis activity of DGAT2 from yeast but not E. coli. The results suggest that rDGAT2 is present as monomer and dimer forms on SDS-PAGE, associated with other proteins, lipids, and membranes, and that post-translational modification of rDGAT2 may be required for its enzymatic activity and/or the E. coli protein is misfolded.

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.

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.

Molecular analysis of a bifunctional fatty acid conjugase/desaturase from tung. Implications for the evolution of plant fatty acid diversity.

The seed oil derived from the tung (Aleurites fordii Hemsl.) tree contains approximately 80% alpha-eleostearic acid (18:3delta(9cis,11trans,13trans)), an unusual conjugated fatty acid that imparts industrially important drying qualities to tung oil. Here, we describe the cloning and functional analysis of two closely related Delta(12) oleate desaturase-like enzymes that constitute consecutive steps in the biosynthetic pathway of eleostearic acid. Polymerase chain reaction screening of a tung seed cDNA library using degenerate oligonucleotide primers resulted in identification of two desaturases, FAD2 and FADX, that shared 73% amino acid identity. Both enzymes were localized to the endoplasmic reticulum of tobacco (Nicotiana tabacum cv Bright-Yellow 2) cells, and reverse transcriptase-polymerase chain reaction revealed that FADX was expressed exclusively within developing tung seeds. Expression of the cDNAs encoding these enzymes in yeast (Saccharomyces cerevisiae) revealed that FAD2 converted oleic acid (18:1delta(9cis)) into linoleic acid (18:2delta(9cis,12cis)) and that FADX converted linoleic acid into alpha-eleostearic acid. Additional characterization revealed that FADX exhibited remarkable enzymatic plasticity, capable of generating a variety of alternative conjugated and delta(12)-desaturated fatty acid products in yeast cells cultured in the presence of exogenously supplied fatty acid substrates. Unlike other desaturases reported to date, the double bond introduced by FADX during fatty acid desaturation was in the trans, rather than cis, configuration. Phylogenetic analysis revealed that tung FADX is grouped with delta(12) fatty acid desaturases and hydroxylases rather than conjugases, which is consistent with its desaturase activity. Comparison of FADX and other lipid-modifying enzymes (desaturase, hydroxylase, epoxygenase, acetylenase, and conjugase) revealed several amino acid positions near the active site that may be important determinants of enzymatic activity.