Overexpression of an isopentenyl diphosphate isomerase gene to enhance trans-polyisoprene production in Eucommia ulmoides Oliver.

BACKGROUND: Natural rubber produced by plants, known as polyisoprene, is the most widely used isoprenoid polymer. Plant polyisoprenes can be classified into two types; cis-polyisoprene and trans-polyisoprene, depending on the type of polymerization of the isoprene unit. More than 2000 species of higher plants produce latex consisting of cis-polyisoprene. Hevea brasiliensis (rubber tree) produces cis-polyisoprene, and is the key source of commercial rubber. In contrast, relatively few plant species produce trans-polyisoprene. Currently, trans-polyisoprene is mainly produced synthetically, and no plant species is used for its commercial production. RESULTS: To develop a plant-based system suitable for large-scale production of trans-polyisoprene, we selected a trans-polyisoprene-producing plant, Eucommia ulmoides Oliver, as the target for genetic transformation. A full-length cDNA (designated as EuIPI, Accession No. AB041629) encoding isopentenyl diphosphate isomerase (IPI) was isolated from E. ulmoides. EuIPI consisted of 1028 bp with a 675-bp open reading frame encoding a protein with 224 amino acid residues. EuIPI shared high identity with other plant IPIs, and the recombinant protein expressed in Escherichia coli showed IPI enzymatic activity in vitro. EuIPI was introduced into E. ulmoides via Agrobacterium-mediated transformation. Transgenic lines of E. ulmoides overexpressing EuIPI showed increased EuIPI expression (up to 19-fold that of the wild-type) and a 3- to 4-fold increase in the total content of trans-polyisoprenes, compared with the wild-type (non-transgenic root line) control. CONCLUSIONS: Increasing the expression level of EuIPI by overexpression increased accumulation of trans-polyisoprenes in transgenic E. ulmoides. IPI catalyzes the conversion of isopentenyl diphosphate to its highly electrophilic isomer, dimethylallyl diphosphate, which is the first step in the biosynthesis of all isoprenoids, including polyisoprene. Our results demonstrated that regulation of IPI expression is a key target for efficient production of trans-polyisoprene in E. ulmoides.

The Ca2+ -dependent DNases are involved in secondary xylem development in Eucommia ulmoides.

Secondary xylem development has long been recognized as a typical case of programmed cell death (PCD) in plants. During PCD, the degradation of genomic DNA is catalyzed by endonucleases. However, to date, no endonuclease has been shown to participate in secondary xylem development. Two novel Ca(2+) -dependent DNase genes, EuCaN1 and EuCaN2, were identified from the differentiating secondary xylem of the tree Eucommia ulmoides Oliv., their functions were studied by DNase activity assay, in situ hybridization, protein immunolocalization and virus-induced gene silencing experiments. Full-length cDNAs of EuCaN1 and EuCaN2 contained an open reading frame of 987 bp, encoding two proteins of 328 amino acids with SNase-like functional domains. The genomic DNA sequence for EuCaN1 had no introns, while EuCaN2 had 8 introns. EuCaN1 and EuCaN2 digested ssDNA and dsDNA with Ca(2+) -dependence at neutral pH. Their expression was confined to differentiating secondary xylem cells and the proteins were localized in the nucleus. Their activity dynamics was closely correlated with secondary xylem development. Secondary xylem cell differentiation is influenced by RNAi of endonuclease genes. The results provide evidence that the Ca(2+) -dependent DNases are involved in secondary xylem development.

Genome-wide identification and expression pattern analysis of the ribonuclease T2 family in Eucommia ulmoides.

The 2',3'-cycling ribonuclease (RNase) genes are catalysts of RNA cleavage and include the RNase T2 gene family. RNase T2 genes perform important roles in plants and have been conserved in the genome of eukaryotic organisms. In this study we identified 21 EURNS genes in Eucommia ulmoides Oliver (E. ulmoides) and analyzed their structure, chromosomal location, phylogenetic tree, gene duplication, stress-related cis-elements, and expression patterns in different tissues. The length of 21 predicted EURNS proteins ranged from 143 to 374 amino acids (aa), their molecular weight (MW) ranged from 16.21 to 42.38 kDa, and their isoelectric point (PI) value ranged from 5.08 to 9.09. Two classifications (class I and class III) were obtained from the conserved domains analysis and phylogenetic tree. EURNS proteins contained a total of 15 motifs. Motif 1, motif 2, motif 3, and motif 7 were distributed in multiple sequences and were similar to the conserved domain of RNase T2. EURNS genes with similar structure and the predicted EURNS proteins with conserved motif compositions are in the same group in the phylogenetic tree. The results of RT-PCR and transcription data showed that EURNS genes have tissue-specific expression and exhibited obvious trends in different developmental stages. Gene duplication analysis results indicated that segment duplication may be the dominant duplication mode in this gene family. This study provides a theoretical basis for research on the RNase T2 gene family and lays a foundation for the further study of EURNS genes.

Structure-function studies of ultrahigh molecular weight isoprenes provide key insights into their biosynthesis.

Some plant trans-1,4-prenyltransferases (TPTs) produce ultrahigh molecular weight trans-1,4-polyisoprene (TPI) with a molecular weight of over 1.0 million. Although plant-derived TPI has been utilized in various industries, its biosynthesis and physiological function(s) are unclear. Here, we identified three novel Eucommia ulmoides TPT isoforms-EuTPT1, 3, and 5, which synthesized TPI in vitro without other components. Crystal structure analysis of EuTPT3 revealed a dimeric architecture with a central hydrophobic tunnel. Mutation of Cys94 and Ala95 on the central hydrophobic tunnel no longer synthesizd TPI, indicating that Cys94 and Ala95 were essential for forming the dimeric architecture of ultralong-chain TPTs and TPI biosynthesis. A spatiotemporal analysis of the physiological function of TPI in E. ulmoides suggested that it is involved in seed development and maturation. Thus, our analysis provides functional and mechanistic insights into TPI biosynthesis and uncovers biological roles of TPI in plants.

Two Eucommia farnesyl diphosphate synthases exhibit distinct enzymatic properties leading to end product preferences.

Farnesyl diphosphate synthase (FPS) is an essential enzyme in the biosynthesis of prenyl precursors for the production of primary and secondary metabolites, including sterols, dolichols, carotenoids and ubiquinones, and for the modification of proteins. Here we identified and characterized two FPSs (EuFPS1 and EuFPS2) from the plant Eucommia ulmoides. The EuFPSs had seven highly conserved prenyltransferase-specific domains that are critical for activity. Complementation and biochemical analyses using bacterially produced recombinant EuFPS isoforms showed that the EuFPSs had FPP synthesis activities both in vivo and in vitro. In addition to the typical reaction mechanisms of FPS, EuFPSs utilized farnesyl diphosphate (FPP) as an allylic substrate and participated in further elongation of the isoprenyl chain, resulting in the synthesis of geranylgeranyl diphosphate. However, despite the high amino acid similarities between the two EuFPS isozymes, their specific activities, substrate preferences, and final reaction products were different. The use of dimethylallyl diphosphate (DMAPP) as an allylic substrate highlighted the differences between the two enzymes: depending on the pH, the metal ion cofactor, and the cofactor concentration, EuFPS2 accumulated geranyl diphosphate as an intermediate product at a constant rate, whereas EuFPS1 synthesized little geranyl diphosphate. The reaction kinetics of the EuFPSs demonstrated that isopentenyl diphosphate and DMAPP were used both as substrates and as inhibitors of EuFPS activity. Taken together, the results indicate that the biosynthesis of FPP is highly regulated by various factors indispensable for EuFPS reactions in plants.

Molecular cloning of a HMG-CoA reductase gene from Eucommia ulmoides Oliver.

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate, which is the first committed step in the pathway for isoprenoid biosynthesis in plants. A full-length cDNA encoding HMGR (designated as EuHMGR, GenBank Accession No. AY796343) was isolated from Eucommia ulmoides by rapid amplification of cDNA ends (RACE). The full-length cDNA of EuHMGR comprises 2281 bp with a 1770-bp open reading frame (ORF) encoding a 590-amino-acid polypeptide with two trans-membrane domains revealed by bioinformatic analysis. Molecular modeling showed that EuHMGR is a new HMGR with a spatial structure similar to other plant HMGRs. The deduced protein has an isoelectric point (pI) of 6.89 and a calculated molecular weight of about 63 kDa. Sequence comparison analysis showed that EuHMGR had highest homology to HMGR from Hevea brasiliensis. As expected, phylogenetic tree analysis indicated that EuHMGR belongs to plant HMGR group. Tissue expression pattern analysis showed that EuHMGR is strongly expressed in the leaves and stems whereas it is only poorly expressed in the roots, which implies that EuHMGR may be a constitutively expressing gene. Functional complementation of EuHMGR in HMGR-deficient mutant yeast JRY2394 demonstrated that EuHMGR mediates the mevalonate biosynthesis in yeast.