Genome sequence of Jatropha curcas L., a non-edible biodiesel plant, provides a resource to improve seed-related traits.

Jatropha curcas (physic nut), a non-edible oilseed crop, represents one of the most promising alternative energy sources due to its high seed oil content, rapid growth and adaptability to various environments. We report ~339 Mbp draft whole genome sequence of J. curcas var. Chai Nat using both the PacBio and Illumina sequencing platforms. We identified and categorized differentially expressed genes related to biosynthesis of lipid and toxic compound among four stages of seed development. Triacylglycerol (TAG), the major component of seed storage oil, is mainly synthesized by phospholipid:diacylglycerol acyltransferase in Jatropha, and continuous high expression of homologs of oleosin over seed development contributes to accumulation of high level of oil in kernels by preventing the breakdown of TAG. A physical cluster of genes for diterpenoid biosynthetic enzymes, including casbene synthases highly responsible for a toxic compound, phorbol ester, in seed cake, was syntenically highly conserved between Jatropha and castor bean. Transcriptomic analysis of female and male flowers revealed the up-regulation of a dozen family of TFs in female flower. Additionally, we constructed a robust species tree enabling estimation of divergence times among nine Jatropha species and five commercial crops in Malpighiales order. Our results will help researchers and breeders increase energy efficiency of this important oil seed crop by improving yield and oil content, and eliminating toxic compound in seed cake for animal feed.

High-titer production of lathyrane diterpenoids from sugar by engineered Saccharomyces cerevisiae.

Euphorbiaceae are an important source of medically important diterpenoids, such as the anticancer drug ingenol-3-angelate and the antiretroviral drug prostratin. However, extraction from the genetically intractable natural producers is often limited by the small quantities produced, while the organic synthesis of terpene-derived drugs is challenging and similarly low-yielding. While transplanting the biosynthetic pathway into a heterologous host has proven successful for some drugs, it has been largely unsuccessful for diterpenoids due to their elaborate biosynthetic pathways and lack of genetic resources and tools for gene discovery. We engineered casbene precursor production in S. cerevisiae, verified the ability of six Euphorbia lathyris and Jatropha curcas cytochrome P450s to oxidize casbene, and optimized the expression of these P450s and an alcohol dehydrogenase to generate jolkinol C, achieving ~800mg/L of jolkinol C and over 1g/L total oxidized casbanes in millititer plates, the highest titer of oxidized diterpenes in yeast reported to date. This strain enables the semisynthesis of biologically active jolkinol C derivatives and will be an important tool in the elucidation of the biosynthetic pathways for ingenanes, tiglianes, and lathyranes. These findings demonstrate the ability of S. cerevisiae to produce oxidized drug precursors in quantities that are sufficient for drug development and pathway discovery.

Engineering low phorbol ester Jatropha curcas seed by intercepting casbene biosynthesis.

KEY MESSAGE: Casbene is a precursor to phorbol esters and down-regulating casbene synthase effectively reduces phorbol ester biosynthesis. Seed-specific reduction of phorbol ester (PE) helps develop Jatropha seed cake for animal nutrition. Phorbol esters (PEs) are diterpenoids present in some Euphorbiaceae family members like Jatropha curcas L. (Jatropha), a tropical shrub yielding high-quality oil suitable as feedstock for biodiesel and bio jet fuel. Jatropha seed contains up to 40 % of oil and can produce oil together with cake containing high-quality proteins. However, skin-irritating and cancer-promoting PEs make Jatropha cake meal unsuitable for animal nutrition and also raise some safety and environmental concerns on its planting and processing. Two casbene synthase gene (JcCASA163 and JcCASD168) homologues were cloned from Jatropha genome and both genes were highly expressed during seed development. In vitro functional analysis proved casbene synthase activity of JcCASA163 in converting geranylgeranyl diphosphate into casbene which has been speculated to be the precursor to PEs. A seed-specific promoter driving inverted repeats for RNAi interference targeting at either JcCASA163 or both genes could effectively down-regulate casbene synthase gene expression with concurrent marked reduction of PE level (by as much as 85 %) in seeds with no pleiotropic effects observed. Such engineered low PE in seed was heritable and co-segregated with the transgene. Our work implicated casbene synthase in Jatropha PE biosynthesis and provided evidence for casbene being the precursor for PEs. The success in reducing seed PE content through down-regulation of casbene synthase demonstrates the feasibility of intercepting PE biosynthesis in Jatropha seed to help address safety concerns on Jatropha plantation and seed processing and facilitate use of its seed protein for animal nutrition.

Improvement on the thermal stability and activity of plant cytosolic ascorbate peroxidase 1 by tailing hyper-acidic fusion partners.

Cytosolic ascorbate peroxidase 1 (APX1) plays a crucial role in regulating the level of plant cellular reactive oxygen species and its thermolability is proposed to cause plant heat-susceptibility. Herein, several hyper-acidic fusion partners, such as the C-terminal peptide tails, were evaluated for their effects on the thermal stability and activity of APX1 from Jatropha curcas and Arabidopsis. The hyper-acidic fusion partners efficiently improved the thermostability and prevented thermal inactivation of APX1 in both plant species with an elevated heat tolerance of at least 2 °C. These hyper-acidified thermostable APX1 fusion variants are of considerable biotechnological potential and can provide a new route to enhance the heat tolerance of plant species especially of inherent thermo-sensitivity.

Long-term chemiluminescence signal is produced in the course of luminol oxidation catalyzed by enhancer-independent peroxidase purified from Jatropha curcas leaves.

Isoenzyme c of horseradish peroxidase (HRP-C) is widely used in enzyme immunoassay combined with chemiluminescence (CL) detection. For this application, HRP-C activity measurement is usually based on luminol oxidation in the presence of hydrogen peroxide (H2O2). However, this catalysis reaction was enhancer dependent. In this study, we demonstrated that Jatropha curcas peroxidase (JcGP1) showed high efficiency in catalyzing luminol oxidation in the presence of H2O2. Compared with HRP-C, the JcGP1-induced reaction was enhancer independent, which made the enzyme-linked immunosorbent assay (ELISA) simpler. In addition, the JcGP1 catalyzed reaction showed a long-term stable CL signal. We optimized the conditions for JcGP1 catalysis and determined the favorable conditions as follows: 50 mM Tris buffer (pH 8.2) containing 10 mM H2 O2, 14 mM luminol and 0.75 M NaCl. The optimum catalysis temperature was 30°C. The detection limit of JcGP1 under optimum condition was 0.2 pM. Long-term stable CL signal combined with enhancer-independent property indicated that JcGP1 might be a valuable candidate peroxidase for clinical diagnosis and enzyme immunoassay with CL detection.

A Cu/Zn superoxide dismutase from Jatropha curcas enhances salt tolerance of Arabidopsis thaliana.

Superoxide dismutases (SODs) are involved in protecting plants against diverse biotic and abiotic stresses. In the present study, a novel Cu/Zn-SOD gene (JcCu/Zn-SOD) was cloned from Jatropha curcas L. Quantitative reverse transcription-polymerase chain reaction analysis revealed that JcCu/Zn-SOD is constitutively expressed in different tissues of J. curcas and induced under NaCl treatment. To characterize the function of this gene with respect to salt tolerance, the construct p35S:JcCu/Zn-SOD was developed and transformed into Arabidopsis using Agrobacterium-mediated transformation. Compared with wild-type, transgenic plants over-expressing JcCu/Zn-SOD showed enhanced tolerance to salt stress during germination, seedling establishment, and growth in terms of longer root, larger rosette area, and a larger number of leaves in addition to higher SOD activity levels under NaCl stress. In addition, over-expression of JcCu/Zn-SOD resulted in lower monodialdehyde content in transgenic Arabidopsis compared to wild-type plants under the same NaCl stress. Therefore, JcCu/Zn-SOD can increase a plant salt stress tolerance potentially by reducing oxidant injury.

Ascorbate peroxidase from Jatropha curcas enhances salt tolerance in transgenic Arabidopsis.

Ascorbate peroxidase (APX) plays a central role in the ascorbate-glutathione cycle and is a key enzyme in cellular H2O2 me-tabolism. It includes a family of isoenzymes with different character-istics, which are identified in many higher plants. In the present study, we isolated the APX gene from Jatropha curcas L, which is similar with other previously characterized APXs as revealed by alignment and phylogenetic analysis of its deduced amino acid sequence. Real-time qPCR analysis showed that the expression level of JcAPX transcript significantly increased under NaCl stress. Subsequently, to elucidate the contribution of JcAPX to the protection against salt-induced oxi-dative stress, the expression construct p35S: JcAPX was created and transformed into Arabidopsis and transcribed. Under 150-mM NaCl stress, compared with wild type (WT), the overexpression of JcAPX in Arabidopsis increased the germination rate, the number of leaves, and the rosette area. In addition, the transgenic plants had longer roots, higher total chlorophyll content, higher total APX activity, and lower H2O2 content than the WT under NaCl stress conditions. These results suggested that higher APX activity in transgenic lines increases the salt tolerance by enhancing scavenging capacity for reactive oxygen spe-cies under NaCl stress conditions.

Lead induced changes in growth and micronutrient uptake of Jatropha curcas L.

Effects of lead treatment on growth and micronutrient uptake in Jatropha curcas L. seedlings were assessed by means of microcosm experiments. Results suggested that superoxide dismutase (SOD) activity increased with increasing lead concentration. There was significant positive correlation between lead treatment concentration and SOD and peroxidase activity. Catalase activity was initiated under lower lead stress but, was inhibited under higher lead exposure. Lead had a stimulating effect on seedlings height and leaf area at lower lead concentrations. The J. curcas can accumulate higher amounts of available lead from soil but can translocate only low amounts to the shoots. Results indicating SOD and peroxidase activity in J. curcas seedlings played an important role in resisting the oxidative stress induced by lead. The addition of lead significantly increased the content of zinc in plant tissue and enhanced the transport of iron from roots to shoots but contributed to a decrease in measured copper, iron, and manganese content.

Cloning, expression, and purification of a Cu/Zn superoxide dismutase from Jatropha curcas.

We report cDNA cloning, expression, purification, and characterization of a novel Cu/ Zn superoxide dismutase (SOD) from Jatropha curcas leaves. The full-length cDNA of this SOD contained a 496-bp open-reading frame (ORF) encoding 162 amino acid residues. The recombinant plasmid containing the SOD coding sequence was introduced into Escherichia coli, and the SOD was expressed as a fusion protein. The recombinant SOD was purified from a high-density fed-batch culture using a combination of immobilized metal ion affinity chromatography (IMAC) and Sephadex G25 desalting chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis indicated that the recombinant SOD was a monomeric protein with a molecular mass of approximately 16.4 kDa. Isoelectric focusing showed that this SOD was a basic protein with pI values of 7.04, 7.33, 8.62, and 8.77. The activity of the SOD was stable at 70 degrees C for 10 min, and in a broad pH range from 4 to 9. The presence of urea (up to 8 M), guanidinium chloride (up to 6 M), and 2-mercaptoethanol (up to 8 mM) had little effect on the activity. The activity decreased gradually with increasing concentrations of imidazole, hydrogen peroxide, and ethylenediaminetetraacetic acid (EDTA). Atomic absorption spectrometry showed the presence of 0.239 copper and 0.258 zinc atoms, respectively, in the SOD polypeptide.

A novel thylakoid ascorbate peroxidase from Jatrophacurcas enhances salt tolerance in transgenic tobacco.

Ascorbate peroxidase (APX) plays an important role in the metabolism of hydrogen peroxide in higher plants. In the present study, a novel APX gene (JctAPX) was cloned from Jatropha curcas L. The deduced amino acid sequence was similar to that of APX of some other plant species. JctAPX has a chloroplast transit peptide and was localized to the chloroplasts by analysis with a JctAPX-green fluorescent protein (GFP) fusion protein. Quantitative polymerase chain reaction (qPCR) analysis showed that JctAPX was constitutively expressed in different tissues from J. curcas and was upregulated by NaCl stress. To characterize its function in salt tolerance, the construct p35S: JctAPX was created and successfully introduced into tobacco by Agrobacterium-mediated transformation. Compared with wild type (WT), the transgenic plants exhibited no morphological abnormalities in the no-stress condition. However, under 200 mM NaCl treatment, JctAPX over-expressing plants showed increased tolerance to salt during seedling establishment and growth. In addition, the transgenic lines showed higher chlorophyll content and APX activity, which resulted in lower H2O2 content than WT when subjected to 400 mM NaCl stress. These results suggest that the increased APX activity in the chloroplasts from transformed plants increased salt tolerance by enhancing reactive oxygen species (ROS)-scavenging capacity under short-term NaCl stress conditions.

Characterization, and expression profile of a phenylalanine ammonia lyase gene from Jatropha curcas L.

A PAL gene designated as JcPAL1 was cloned from J. curcas L. The full-length is 2336 bp in size with one intron and two exons, encoding a polypeptide of 713 amino acids. Its 5'-upstream region is rich in putative cis-elements including not only PAL typical TATA box, L-box and transcriptional initiation site (TIS) but also light responding motifs. Expression pattern analysis indicated that JcPAL1 were expressed in all tissues, most highly in flowers. When Treated with ABA, GA3, high and low temperature, expression of JcPAL1 were induced. Recombinant JcPAL1 has a pH optimum at 8.7 and a temperature optimum at 60°C in 100 mM Tris-HCl buffer. The Km and Kcat values are 0.125 mM and 1.73 S(-1) for L: -phenylalanine, and 1.312 mM and 0.109 S(-1) for L: -tyrosine, respectively. These findings suggested that JcPAL1 might involve in the J. curcas responding to various stresses and L: -Phe should be its true physiological substrate. This study is essential prior to uncover whether and how the PAL initiated phenylpropanoid metabolic networks functioning in the defense responses of J. curcas.

Cloning, overexpression, purification, and characterization of a new iron superoxide dismutase from Jatropha curcas.

A novel iron superoxide dismutase (Fe-SOD) gene from Jatropha curcas was cloned and expressed in Escherichia coli BL21 (DE3). This recombinant enzyme was isolated by a combined procedure involving immobilized metal-ion affinity chromatography and ion-exchange chromatography. The apparent molecular mass of this purified enzyme (designated as JcFe-SOD) was 35 kDa on SDS-PAGE. The full-length complementary DNA sequence of JcFe-SOD contained a 918-bp open reading frame encoding a 305-amino-acid precursor of 34.589 kDa. The result of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry showed that the purified enzyme may own two forms: a dimer and a monomer. The enzyme was relatively stable and showed 54% activity when incubated in 70°C for 60 Min. JcFe-SOD was found to have good pH stability in the pH range of 5.5-9.5 at 25°C over 1 H incubation. The activity of this enzyme was gradually inhibited by increasing concentration of H2O2, 2-mercaptoethanol, and ethylenediaminetetraacetic acid. An assay of the atomic absorption spectrum showed the presence of 0.41 atom of Fe in each SOD subunit.

Revisiting Jatropha curcas Monomeric Esterase: A Dienelactone Hydrolase Compatible with the Electrostatic Catapult Model.

Jatropha curcas contains seeds with a high oil content, suitable for biodiesel production. After oil extraction, the remaining mass can be a rich source of enzymes. However, data from the literature describing physicochemical characteristics for a monomeric esterase from the J. curcas seed did not fit the electrostatic catapult model for esterases/lipases. We decided to reevaluate this J. curcas esterase and extend its characterization to check this apparent discrepancy and gain insights into the enzyme's potential as a biocatalyst. After anion exchange chromatography and two-dimensional gel electrophoresis, we identified the enzyme as belonging to the dienelactone hydrolase family, characterized by a cysteine as the nucleophile in the catalytic triad. The enzyme displayed a basic optimum hydrolysis pH of 9.0 and an acidic pI range, in contrast to literature data, making it well in line with the electrostatic catapult model. Furthermore, the enzyme showed low hydrolysis activity in an organic solvent-containing medium (isopropanol, acetonitrile, and ethanol), which reverted when recovering in an aqueous reaction mixture. This enzyme can be a valuable tool for hydrolysis reactions of short-chain esters, useful for pharmaceutical intermediates synthesis, due to both its high hydrolytic rate in basic pH and its stability in an organic solvent.

Molecular cloning and characterization of phospholipase D from Jatropha curcas.

Phospholipase D (PLD, EC 3.1.4.4) is a key enzyme involved in phospholipid catabolism, initiating a lipolytic cascade in membrane deterioration during senescence and stress, which was cloned from Jatropha curcas L., an important plant species as its seed is the raw material for biodiesels. The cDNA was 2,886 bp in length with a complete open reading frame of 2,427 bp which encoded a polypeptide of 808 amino acids including a putative signal peptide of 53 amino acid residues and a mature protein of 755 amino acids with a predicted molecular mass of 86 kD and a pI of 5.44, having two highly conserved HKD' motifs. Phylogenetic analysis indicated the J. curcas PLD alpha (JcPLDalpha) showed a high similarity to other PLD alpha from plants. Semi-quantitative RT-PCR analysis revealed that it was especially abundant in root, stem, leaf, endosperm and flower, weakly in seed. And the JcPLDalpha was increasedly expressed in leaf undergoing environmental stress such as salt (300 mM NaCl), drought (30% PEG), cold (4degreeC) and heat (50degreeC). The JcPLDalpha protein was successfully expressed in Escherichia coli and showed high enzymatic activities. Maximal activity was at pH 8 and 60degreeC.

Cloning and expression analysis of carboxyltransferase of acetyl-coA carboxylase from Jatropha curcas.

A full-length cDNA of the carboxyltransferase (accA) gene of acetyl-coenzym A (acetyl-CoA) carboxylase from Jatropha curcas was cloned and sequenced. The gene with an open reading frame (ORF) of 1149 bp encodes a polypeptide of 383 amino acids, with a molecular mass of 41.9 kDa. Utilizing fluorogenic real-time polymerase chain reaction (RT-PCR), the expression levels of the accA gene in leaves and fruits at early, middle and late stages under pH 7.0/8.0 and light/darkness stress were investigated. The expression levels of the accA gene in leaves at early, middle and late stages increased significantly under pH 8.0 stress compared to pH 7.0. Similarly, the expression levels in fruits showed a significant increase under darkness condition compared to the control. Under light stress, the expression levels in the fruits at early, middle and late stages showed the largest fluctuations compared to those of the control. These findings suggested that the expression levels of the accA gene are closely related to the growth conditions and developmental stages in the leaves and fruits of Jatropha curcas.

Cloning and functional characterization of an acyl-acyl carrier protein thioesterase (JcFATB1) from Jatropha curcas.

A full-length cDNA of an acyl-acyl carrier protein (ACP) thioesterase (TE) (EC 3.1.2.14), named JcFATB1, was isolated from the woody oil plant Jatropha curcas L. The deduced amino acid sequence of the cDNA shares about 78% identity with FATB TEs, but only about 33% identity with FATA TEs from other plants. The deduced sequence also contains two essential residues (H(317) and C(352)) for TE catalytic activity and a putative chloroplast transit peptide at the N-terminal. Southern blot analysis revealed that a single copy of JcFATB1 is present in the J. curcas genome, and semi-quantitative PCR analysis showed that JcFATB1 was expressed in all tissues that were examined, most strongly in seeds, in which its expression peaked in late developmental stages. Seed-specific overexpression of the JcFATB1 cDNA in Arabidopsis resulted in increased levels of saturated fatty acids, especially palmitate, and in reduced levels of unsaturated fatty acids. The findings suggest that JcFATB1 from this woody oil plant can function as a saturated acyl-ACP TE and could potentially modify the seed oil of J. curcas to increase its levels of palmitate.

Growth and antioxidant responses in Jatropha curcas cotyledons under lead stress.

Jatropha curcas embryos were grown in vitro to observe the effects of lead on cotyledon responses. The cotyledon biomass increased initially and then decreased with increasing lead concentration. The SOD activity increased gradually up to 200 microM and then decreased. The POD activity showed a similar trend. The CAT activity was increased at all lead concentrations, the highest activity being observed at 200 microM. However, the PAL activity was inhibited significantly except for 100 microM. Anaylsis by electrophoresis suggested a significant correlation between lead concentration and patterns of SOD, POD and CAT isoenzymes, and these results were consistent with changes of the antioxidant enzyme activities as assayed in solution.

Aluminum directly inhibits alternative oxidase pathway and changes metabolic and redox parameters on Jatropha curcas cell culture.

Aluminum (Al) toxicity has been recognized to be a main limiting factor of crop productivity in acid soil. Al interacts with cell walls disrupting the functions of the plasma membrane and is associated with oxidative damage and mitochondrial dysfunction. Jatropha curcas L. (J. curcas) is a drought resistant plant, widely distributed around the world, with great economic and medicinal importance. Here we investigated the effects of Al on J. curcas mitochondrial function and cell viability, analyzing mitochondrial respiration, phenolic compounds, reducing sugars and cell viability in cultured J. curcas cells. The results showed that at 70 µM, Al limited mitochondrial respiration by inhibiting the alternative oxidase (AOX) pathway in the respiratory chain. An increased concentration of reducing sugars and reduced concentration of intracellular phenolic compounds was observed during respiratory inhibition. After inhibition, a time-dependent upregulation of AOX mRNA was observed followed by restoration of respiratory activity and reducing sugar concentrations. Cultured J. curcas cells were very resistant to Al-induced cell death. In addition, at 70 µM, Al also appeared as an inhibitor of cell wall invertase. In conclusion, Al tolerance in cultured J. curcas cells involves a inhibition of mitochondrial AOX pathway, which seems to start an oxidative burst to induce AOX upregulation, which in turn restores consumption of O2 and substrates. These data provide new insight into the signaling cascades that modulate the Al tolerance mechanism.

Molecular cloning and expression analysis of a gene encoding a putative beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III) from Jatropha curcas.

A cDNA clone encoding a putative beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III) was isolated from Jatropha curcas L., a woody oil plant. The cDNA clone (named JcKAS III) contained a 1203-bp open reading frame coding for 400 amino acids with a predicted molecular mass of about 42 kDa. The deduced amino acid sequence of the cDNA clone shares about 80% identity to KAS III from other plants, and contains a conserved Cys(176) in the active site and the amino acid motif G(355)NTSAAS(361) which is responsible for binding regulatory acyl-ACPs. Southern blotting analysis indicated that JcKAS III is a single copy gene in the J. curcas genome. Quantitative real-time PCR analysis showed that JcKAS III was expressed in all tissues examined with highest expression in roots, and that expression of JcKAS III increased as seeds developed.

Genome-wide comparative analysis of papain-like cysteine protease family genes in castor bean and physic nut.

Papain-like cysteine proteases (PLCPs) are a class of proteolytic enzymes involved in many plant processes. Compared with the extensive research in Arabidopsis thaliana, little is known in castor bean (Ricinus communis) and physic nut (Jatropha curcas), two Euphorbiaceous plants without any recent whole-genome duplication. In this study, a total of 26 or 23 PLCP genes were identified from the genomes of castor bean and physic nut respectively, which can be divided into nine subfamilies based on the phylogenetic analysis: RD21, CEP, XCP, XBCP3, THI, SAG12, RD19, ALP and CTB. Although most of them harbor orthologs in Arabidopsis, several members in subfamilies RD21, CEP, XBCP3 and SAG12 form new groups or subgroups as observed in other species, suggesting specific gene loss occurred in Arabidopsis. Recent gene duplicates were also identified in these two species, but they are limited to the SAG12 subfamily and were all derived from local duplication. Expression profiling revealed diverse patterns of different family members over various tissues. Furthermore, the evolution characteristics of PLCP genes were also compared and discussed. Our findings provide a useful reference to characterize PLCP genes and investigate the family evolution in Euphorbiaceae and species beyond.