Untargeted Metabolomic Analysis of Leaves and Roots of Jatropha curcas Genotypes with Contrasting Levels of Phorbol Esters.

AIMS: Phorbol esters (PE) are toxic diterpenoids accumulated in physic nut (Jatropha curcas L.) seed tissues. Their biosynthetic pathway remains unknown, and the participation of roots in this process may be possible. Thus, we set out to study the deposition pattern of PE and other terpenoids in roots and leaves of genotypes with detected (DPE) and not detected (NPE) phorbol esters based on previous studies. OUTLINE OF DATA RESOURCES: We analyzed physic nut leaf and root organic extracts using LC-HRMS. By an untargeted metabolomics approach, it was possible to annotate 496 and 146 metabolites in the positive and negative electrospray ionization modes, respectively. KEY RESULTS: PE were detected only in samples of the DPE genotype. Remarkably, PE were found in both leaves and roots, making this study the first report of PE in J. curcas roots. Furthermore, untargeted metabolomic analysis revealed that diterpenoids and apocarotenoids are preferentially accumulated in the DPE genotype in comparison with NPE, which may be linked to the divergence between the genotypes concerning PE biosynthesis, since sesquiterpenoids showed greater abundance in the NPE. UTILITY OF THE RESOURCE: The LC-HRMS files, publicly available in the MassIVE database (identifier MSV000092920), are valuable as they expand our understanding of PE biosynthesis, which can assist in the development of molecular strategies to reduce PE levels in toxic genotypes, making possible the food use of the seedcake, as well as its potential to contain high-quality spectral information about several other metabolites that may possess biological activity.

Functional Characterization of JcSWEET12 and JcSWEET17a from Physic Nut.

Physic nut (Jatropha curcas L.) has attracted extensive attention because of its fast growth, easy reproduction, tolerance to barren conditions, and high oil content of seeds. SWEET (Sugar Will Eventually be Exported Transporter) family genes contribute to regulating the distribution of carbohydrates in plants and have great potential in improving yield and stress tolerance. In this study, we performed a functional analysis of the homology of these genes from physic nut, JcSWEET12 and JcSWEET17a. Subcellular localization indicated that the JcSWEET12 protein is localized on the plasma membrane and the JcSWEET17a protein on the vacuolar membrane. The overexpression of JcSWEET12 (OE12) and JcSWEET17a (OE17a) in Arabidopsis leads to late and early flowering, respectively, compared to the wild-type plants. The transgenic OE12 seedlings, but not OE17a, exhibit increased salt tolerance. In addition, OE12 plants attain greater plant height and greater shoot dry weight than the wild-type plants at maturity. Together, our results indicate that JcSWEET12 and JcSWEET17a play different roles in the regulation of flowering time and salt stress response, providing a novel genetic resource for future improvement in physic nut and other plants.

The study on interacting factors and functions of GASA6 in Jatropha curcas L.

BACKGROUND: The gibberellic acid-stimulated Arabidopsis (GASA) gene encodes a class of cysteine-rich functional proteins and is ubiquitous in plants. Most GASA proteins are influence the signal transmission of plant hormones and regulate plant growth and development, however, their function in Jatropha curcas is still unknown. RESULTS: In this study, we cloned JcGASA6, a member of the GASA family, from J. curcas. The JcGASA6 protein has a GASA-conserved domain and is located in the tonoplast. The three-dimensional structure of the JcGASA6 protein is highly consistent with the antibacterial protein Snakin-1. Additionally, the results of the yeast one-hybrid (Y1H) assay showed that JcGASA6 was activated by JcERF1, JcPYL9, and JcFLX. The results of the Y2H assay showed that both JcCNR8 and JcSIZ1 could interact with JcGASA6 in the nucleus. The expression of JcGASA6 increased continuously during male flower development, and the overexpression of JcGASA6 was associated with filament elongation of the stamens in tobacco. CONCLUSION: JcGASA6, a member of the GASA family in J. curcas, play an important role in growth regulation and floral development (especially in male flower). It is also involved in the signal transduction of hormones, such as ABA, ET, GA, BR, and SA. Also, JcGASA6 is a potential antimicrobial protein determined by its three-dimensional structure.

Reversible down-regulation of photosystems I and II leads to fast photosynthesis recovery after long-term drought in Jatropha curcas.

Jatropha curcas is a drought-tolerant plant that maintains its photosynthetic pigments under prolonged drought, and quickly regains its photosynthetic capacity when water is available. It has been reported that drought stress leads to increased thermal dissipation in PSII, but that of PSI has been barely investigated, perhaps due to technical limitations in measuring the PSI absolute quantum yield. In this study, we combined biochemical analysis and spectroscopic measurements using an integrating sphere, and verified that the quantum yields of both photosystems are temporarily down-regulated under drought. We found that the decrease in the quantum yield of PSII was accompanied by a decrease in the core complexes of PSII while light-harvesting complexes are maintained under drought. In addition, in drought-treated plants, we observed a decrease in the absolute quantum yield of PSI as compared with the well-watered control, while the amount of PSI did not change, indicating that non-photochemical quenching occurs in PSI. The down-regulation of both photosystems was quickly lifted in a few days upon re-watering. Our results indicate, that in J. curcas under drought, the down-regulation of both PSII and PSI quantum yield protects the photosynthetic machinery from uncontrolled photodamage.

Identification and promoter analysis of a GA-stimulated transcript 1 gene from Jatropha curcas.

KEY MESSAGE: Overexpression of JcGAST1 promotes plant growth but inhibits pistil development. The pyrimidine box and CGTCA motif of the JcGAST1 promoter were responsible for the GA and MeJA responses. Members of the gibberellic acid-stimulated Arabidopsis (GASA) gene family play roles in plant growth and development, particularly in flower induction and seed development. However, there is still relatively limited knowledge of GASA genes in Jatropha curcas. Herein, we identified a GASA family gene from Jatropha curcas, namely, JcGAST1, which encodes a protein containing a conserved GASA domain. Sequence alignment showed that the JcGAST1 protein shares 76% sequence identity and 80% sequence similarity with SlGAST1. JcGAST1 had higher expression and protein levels in the female flowers than in the male flowers. Overexpression of JcGAST1 in tobacco promotes plant growth but inhibits pistil development. JcGAST1 expression was upregulated by GA and downregulated by MeJA. Promoter analysis indicated that the pyrimidine box and CGTCA motif were the GA- and MeJA-responsive elements of the JcGAST1 promoter. Using a Y1H screen, six transcription factors were found to interact with the pyrimidine box, and three transcription factors were found to interact with the CGTCA motif. Overall, the results of this study improve our understanding of the JcGAST1 gene and provide useful information for further studies.

An ortholog of the MADS-box gene SEPALLATA3 regulates stamen development in the woody plant Jatropha curcas.

MAIN CONCLUSION: Overexpression of JcSEP3 causes defective stamen development in Jatropha curcas, in which brassinosteroid and gibberellin signaling pathways may be involved. SEPALLATAs (SEPs), the class E genes of the ABCE model, are required for floral organ determination. In this study, we investigated the role of the JcSEP3 gene in floral organ development in the woody plant Jatropha curcas. Transgenic Jatropha plants overexpressing JcSEP3 displayed abnormal phenotypes such as deficient anthers and pollen, as well as free stamen filaments, whereas JcSEP3-RNA interference (RNAi) transgenic plants had no obvious phenotypic changes, suggesting that JcSEP3 is redundant with other JcSEP genes in Jatropha. Moreover, we compared the transcriptomes of wild-type plants, JcSEP3-overexpressing, and JcSEP3-RNAi transgenic plants. In the JcSEP3-overexpressing transgenic plants, we discovered 25 upregulated genes involved in anther and pollen development, as well as 12 induced genes in brassinosteroid (BR) and gibberellin (GA) signaling pathways. These results suggest that JcSEP3 directly or indirectly regulates stamen development, concomitant with the regulation of BR and GA signaling pathways. Our findings help to understand the roles of SEP genes in stamen development in perennial woody plants.

Rapid detoxification of Jatropha curcas cake by fermentation with a combination of three microbial strains and characterization of their metabolic profiles.

AIM: Our previous study reported a strain that can detoxify Jatropha curcas L. cake (JCC), but the detoxification duration is long. This study intends to explore the efficient detoxification of JCC through multi-strain collaborative fermentation to accelerate the detoxification process. METHODS AND RESULTS: Mucor circinelloides SCYA25 strain that we previously reported can effectively degrade the toxicity of JCC, and the newly screened Bacillus megaterium SCYA10 and Geotrichum candidum SCYA23 strains were used to detoxify JCC. Different solid-state-fermentation (SSF) parameters were optimized by single-factor tests and response surface methodology. A detoxification rate established by zebrafish toxicity of JCC at 96% was achieved under the following optimized conditions: the combination ratio of B. megaterium SCYA10, G. candidum SCYA23 and M. circinelloides SCYA25 at 2:3:1, a total injection amount of 15.25%, a feed to water ratio of 1:0.68, a fermentation temperature of 30.3°C and fermentation duration of 21.5 days. The protein content of fermented JCC (FJCC) increased, while the concentrations of ether extract, crude fibre and toxins were all degraded considerably. Metabolomics analysis revealed that the fermentation increased the contents of neurotransmitter receptor modulator, emulsifier, aromatic substances and insecticidal compounds, as well as decreasing the contents of oxidative stress and neurotoxic substances. A rat feeding trial showed that the growth performance of the rats provided with the FJCC diet was similar to that of the corn-soybean meal group, and no lesions in the liver and kidney were observed. CONCLUSION: The co-bio-fermentation process can effectively detoxify JCC and improve its nutritional value, which means it could be served as a protein feed in animal husbandry. SIGNIFICANCE: The combination of three microbial strains can detoxify JCC in a safe and effective manner to provide a great potential alternative to soybean meal. The research also suggests that metabonomics and bioinformatics are useful tools for revealing the bio-detoxification mechanism.

Heterologous Expression of Jatropha curcas Fatty Acyl-ACP Thioesterase A (JcFATA) and B (JcFATB) Affects Fatty Acid Accumulation and Promotes Plant Growth and Development in Arabidopsis.

Plant fatty acyl-acyl carrier protein (ACP) thioesterases terminate the process of de novo fatty acid biosynthesis in plastids by hydrolyzing the acyl-ACP intermediates, and determine the chain length and levels of free fatty acids. They are of interest due to their roles in fatty acid synthesis and their potential to modify plant seed oils through biotechnology. Fatty acyl-ACP thioesterases (FAT) are divided into two families, i.e., FATA and FATB, according to their amino acid sequence and substrate specificity. The high oil content in Jatropha curcas L. seed has attracted global attention due to its potential for the production of biodiesel. However, the detailed effects of JcFATA and JcFATB on fatty acid biosynthesis and plant growth and development are still unclear. In this study, we found that JcFATB transcripts were detected in all tissues and organs examined, with especially high accumulation in the roots, leaves, flowers, and some stages of developing seeds, and JcFATA showed a very similar expression pattern. Subcellular localization of the JcFATA-GFP and JcFATB-GFP fusion protein in Arabidopsis leaf protoplasts showed that both JcFATA and JcFATB localized in chloroplasts. Heterologous expression of JcFATA and JcFATB in Arabidopsis thaliana individually generated transgenic plants with longer roots, stems and siliques, larger rosette leaves, and bigger seeds compared with those of the wild type, indicating the overall promotion effects of JcFATA and JcFATB on plant growth and development while JcFATB had a larger impact. Compositional analysis of seed oil revealed that all fatty acids except 22:0 were significantly increased in the mature seeds of JcFATA-transgenic Arabidopsis lines, especially unsaturated fatty acids, such as the predominant fatty acids of seed oil, 18:1, 18:2, and 18:3. In the mature seeds of the JcFATB-transgenic Arabidopsis lines, most fatty acids were increased compared with those in wild type too, especially saturated fatty acids, such as 16:0, 18:0, 20:0, and 22:0. Our results demonstrated the promotion effect of JcFATA and JcFATB on plant growth and development, and their possible utilization to modify the seed oil composition and content in higher plants.

Model-assisted identification of metabolic engineering strategies for Jatropha curcas lipid pathways.

Efficient approaches to increase plant lipid production are necessary to meet current industrial demands for this important resource. While Jatropha curcas cell culture can be used for in vitro lipid production, scaling up the system for industrial applications requires an understanding of how growth conditions affect lipid metabolism and yield. Here we present a bottom-up metabolic reconstruction of J. curcas supported with labeling experiments and biomass characterization under three growth conditions. We show that the metabolic model can accurately predict growth and distribution of fluxes in cell cultures and use these findings to pinpoint energy expenditures that affect lipid biosynthesis and metabolism. In addition, by using constraint-based modeling approaches we identify network reactions whose joint manipulation optimizes lipid production. The proposed model and computational analyses provide a stepping stone for future rational optimization of other agronomically relevant traits in J. curcas.

Bio-detoxification of Jatropha curcas L. cake by a soil-borne Mucor circinelloides strain using a zebrafish survival model and solid-state fermentation.

AIMS: The aims of the study were to (i) improve the evaluation criteria of detoxifying Jatropha curcas L. cake (JCC), (ii) isolate and characterize a JCC tolerant strain, (iii) explore its JCC detoxifying potential. METHODS AND RESULTS: The zebrafish was employed as a survival model to screen the strains capable of detoxifying JCC. A strain identified as Mucor circinelloides SCYA25, which is highly capable of degrading all toxic components, was isolated from soil. Different solid-state fermentation parameters were optimized by response surface methodology. The optimal values for inoculation amount, moisture content, temperature, and time were found to be 18% (1·8 × 106 spores g-1 cake), 66%, 26, and 36 days, respectively, to achieve maximum detoxification of the JCC (92%). Under optimal fermentation conditions, the protein content of JCC was increased, while the concentrations of ether extract, crude fiber, toxins, and anti-nutritional substances were all degraded considerably (P < 0·05). Scanning electron microscopy and Fourier transform infrared spectrometer analysis revealed that the fermentation process could disrupt the surface structure and improve the ratio of α-helix to ß-folding in the JCC protein, which may improve the digestibility when the detoxified JCC is used as a feedstuff. CONCLUSIONS: Our results indicate that M. circinelloides SCYA25 is able to detoxify JCC and improve its nutritional profile, which is beneficial to the safe utilization of JCC as a protein feedstuff. SIGNIFICANCE AND IMPACT OF THE STUDY: The newly identified M. circinelloides SCYA25 detoxified JCC in a safe manner to provide a potential alternative to soybean meal for the feed industry. These results also provide a new perspective and method for the toxicity evaluation and utilization of JCC and similar toxic agricultural by-products.

The Occurrence of Triple Catalytic Characteristics of Yeast Lipases and Their Application Prospects in Biodiesel Production from Non-Edible Jatropha curcas Oil in a Solvent-Free System.

Extracellular and cell-bound lipase-producing yeasts were isolated from the palm oil mill wastes and investigated for their potential uses as biocatalysts in biodiesel production. Twenty-six yeast strains were qualitatively screened as lipase producers. From those yeast strains, only six were selected and screened further for quantitative lipase production.The phylogenetic affiliations of the yeast strains were confirmed by investigating the D1/D2 domains of 26S rDNA and ITS1-5.8S-ITS2 molecular regions of the six yeast strains selected as potent lipase producers. The three yeast strains A4C, 18B, and 10F showed a close association with Magnusiomyces capitatus. Two yeast strains (17B and AgB) had a close relationship with Saprochaete clavata, whereas the strain AW2 was identified as Magnusiomyces spicifer. Three main catalytic activities of the yeast lipases were evaluated and Magnusiomyces capitatus A4C, among the selected lipase-producing yeasts, had the highest extracellular lipolytic enzyme activity (969 U/L) with the cell-bound lipolytic enzyme activity of 11.3 U/gdm. The maximum cell-bound lipolytic activity (12.4 U/gdm) was observed in the cell-bound lipase fraction produced by Magnusiomyces spicifer AW2 with an extracellular lipolytic enzyme activity of 886 U/L. Based on the specific hydrolytic enzymatic activities, the cell-bound lipases (CBLs) from the three yeast strains M. capitatus A4C, M. spicifer AW2, and Saprochaete clavata 17B were further investigated for biodiesel production. Among them, the CBL from M. spicifer AW2 synthesized the most FAME (fatty acid methyl esters) at 81.2% within 12 h indicating that it has potential for application in enzymatic biodiesel production.

Detoxification of jatropha kernel meal to utilize it as aqua-feed.

BACKGROUND: Jatropha is an oilseed crop with high kernel oil (55-58%) and protein (26-29%) contents, which makes it a good source of biodiesel and animal/aqua-feed. However, the presence of anti-nutritional toxins, such as phorbol esters, lectins, trypsin inhibitor, phytate, and saponins, restricts its use as feed. This paper describes chemical, ultraviolet (UV) radiation, and biological treatments for detoxification of jatropha kernel meal. Raw, defatted, and one-time and two-times mechanically expressed oil samples were analyzed for toxins. Chemical treatment involved heating with 90% methanol and 4% sodium hydroxide. UV treatment was carried out at UV light intensity of 53.4 mW cm-2 for 30 min. For biological treatment, cell-free extract from Pseudomonas aeruginosa (strain PAO1) was mixed with kernel meal for detoxification. RESULTS: Among treatments, chemical treatment was most effective in reducing all toxins, with phorbol esters in the range 0.034-0.052 mg g-1 , lectin 0.082-10.766 mg g-1 , trypsin inhibitor 10.499-11.350 mg g-1 , phytate 2.475-5.769 mg g-1 , and saponins 0.044-0.098 mg g-1 . Biological treatment reduced all toxins except phytate, whereas UV treatment could not reduce any of toxins and, hence, cannot be used for aqua-feed preparation. Pellets prepared from chemically detoxified kernel meal with the least oil content (defatted) resulted in the highest strength (70.93 N). CONCLUSION: Chemically treated jatropha kernel meal can be used for aqua-feed pellet preparation because of its low toxin content. The highest compressive strength was obtained for pellets with the least oil content (defatted). Biological treatment time must have been extended for many hours instead of 24 h. Jatropha kernel meal treated chemically can be recommended for aqua-feed manufacturing. © 2021 Society of Chemical Industry.

Quantitative Proteome Analysis of Jatropha curcas L. Genotypes with Contrasting Levels of Phorbol Esters.

The phorbol esters in the seeds of Jatropha curcas are a major hindrance to the full exploitation of the potential of this oil crop as a source of raw material for the production of biodiesel. Here, various quantitative proteomic strategies are used to establish the proteomes of roots, leaves, and endosperm of two genotypes of J. curcas with contrasting levels of phorbol esters in the seeds. In total 4532, 1775, and 503 proteins are identified respectively in roots, leaves, and endosperm, comprising 5068 unique proteins; of this total, 185 are differentially abundant in roots, 72 in leaves, and 20 in the endosperm. The biosynthetic pathways for flavonoids and terpenoids are well represented in roots, including the complete set of proteins for the mevalonate and non-mevalonate/Deoxyxylulose 5-Phosphate pathways, and proteins involved in the branches which lead to the synthesis tricyclic diterpenoids and gibberellins. Also, casbene synthase which catalyzes the first committed step in the biosynthesis of tigliane-type diterpenes is identified in roots of both genotypes, but not in leaves and endosperm. This dataset will be a valuable resource to explore the biochemical basis of the low toxicity of Jatropha genotypes with low concentration of phorbol esters in the seeds.

Towards Thermally Reversible Networks Based on Furan-Functionalization of Jatropha Oil.

A novel biobased monomer for the preparation of thermally reversible networks based on the Diels-Alder reaction was synthesized from jatropha oil. The oil was epoxidized and subsequently reacted with furfurylamine to attach furan groups via an epoxide ring opening reaction. However, furfurylamine also reacted with the ester groups of the triglycerides via aminolysis, thus resulting in short-chain molecules that ultimately yielded brittle thermally reversible polymers upon cross-linking via a Diels-Alder reaction. A full-factorial experimental design was used in finding the optimum conditions to minimize ester aminolysis and to maximize the epoxide ring opening reaction as well as the number of furans attached to the modified oil. The optimum conditions were determined experimentally and were found to be 80 °C, 24 h, 1:1 molar ratio, with 50 mol % of LiBr with respect to the modified oil, resulting in 35% of ester conversion, 99% of epoxide conversion, and an average of 1.32 furans/triglyceride. Ultimately, further optimization by a statistical approach led to an average of 2.19 furans per triglyceride, which eventually yielded a flexible network upon cross-linking via a Diels-Alder reaction instead of the brittle one obtained when the furan-functionalization reaction was not optimized.

JCDB: a comprehensive knowledge base for Jatropha curcas, an emerging model for woody energy plants.

BACKGROUND: Jatropha curcas is an oil-bearing plant, and has seeds with high oil content (~ 40%). Several advantages, such as easy genetic transformation and short generation duration, have led to the emergence of J. curcas as a model for woody energy plants. With the development of high-throughput sequencing, the genome of Jatropha curcas has been sequenced by different groups and a mass of transcriptome data was released. How to integrate and analyze these omics data is crucial for functional genomics research on J. curcas. RESULTS: By establishing pipelines for processing novel gene identification, gene function annotation, and gene network construction, we systematically integrated and analyzed a series of J. curcas transcriptome data. Based on these data, we constructed a J. curcas database (JCDB), which not only includes general gene information, gene functional annotation, gene interaction networks, and gene expression matrices but also provides tools for browsing, searching, and downloading data, as well as online BLAST, the JBrowse genome browser, ID conversion, heatmaps, and gene network analysis tools. CONCLUSIONS: JCDB is the most comprehensive and well annotated knowledge base for J. curcas. We believe it will make a valuable contribution to the functional genomics study of J. curcas. The database is accessible at http://jcdb.xtbg.ac.cn.

Genome-wide identification and expression profile of HD-ZIP genes in physic nut and functional analysis of the JcHDZ16 gene in transgenic rice.

BACKGROUND: Homeodomain-leucine zipper (HD-ZIP) transcription factors play important roles in the growth, development and stress responses of plants, including (presumably) physic nut (Jatropha curcas), which has high drought and salinity tolerance. However, although physic nut's genome has been released, there is little knowledge of the functions, expression profiles and evolutionary histories of the species' HD-ZIP genes. RESULTS: In this study, 32 HD-ZIP genes were identified in the physic nut genome (JcHDZs) and divided into four groups (I-IV) based on phylogenetic analysis with homologs from rice, maize and Arabidopsis. The analysis also showed that most of the JcHDZ genes were closer to members from Arabidopsis than to members from rice and maize. Of the 32 JcHDZ genes, most showed differential expression patterns among four tissues (root, stem cortex, leaf, and seed). Expression profile analysis based on RNA-seq data indicated that 15 of the JcHDZ genes respond to at least one abiotic stressor (drought and/or salinity) in leaves at least at one time point. Transient expression of a JcHDZ16-YFP fusion protein in Arabidopsis protoplasts cells showed that JcHDZ16 is localized in the nucleus. In addition, rice seedlings transgenically expressing JcHDZ16 had lower proline contents and activities of antioxidant enzymes (catalase and superoxide dismutase) together with higher relative electrolyte leakage and malondialdehyde contents under salt stress conditions (indicating higher sensitivity) than wild-type plants. The transgenic seedlings also showed increased sensitivity to exogenous ABA, and increases in the transcriptional abundance of several salt stress-responsive genes were impaired in their responses to salt stress. Further data on JcHDZ16-overexpressing plants subjected to salt stress treatment verified the putative role of JcHDZ genes in salt stress responses. CONCLUSION: Our results may provide foundations for further investigation of functions of JcHDZ genes in responses to abiotic stress, and promote application of JcHDZ genes in physic nut breeding.

The regulation of P700 is an important photoprotective mechanism to NaCl-salinity in Jatropha curcas.

Salinity commonly affects photosynthesis and crop production worldwide. Salt stress disrupts the fine balance between photosynthetic electron transport and the Calvin cycle reactions, leading to over-reduction and excess energy within the thylakoids. The excess energy triggers reactive oxygen species (ROS) overproduction that causes photoinhibition in both photosystems (PS) I and II. However, the role of PSI photoinhibition and its physiological mechanisms for photoprotection have not yet been fully elucidated. In the present study, we analyzed the effects of 15 consecutive days of 100 mM NaCl in Jatropha curcas plants, primarily focusing on the photosynthetic electron flow at PSI level. We found that J. curcas plants have important photoprotective mechanisms to cope with the harmful effects of salinity. We show that maintaining P700 in an oxidized state is an important photoprotector mechanism, avoiding ROS burst in J. curcas exposed to salinity. In addition, upon photoinhibition of PSI, the highly reduced electron transport chain triggers a significant increase in H2 O2 content which can lead to the production of hydroxyl radical by Mehler reactions in chloroplast, thereby increasing PSI photoinhibition.

Genome-wide screening of hexokinase gene family and functional elucidation of HXK2 response to cold stress in Jatropha curcas.

Hexokinase, the key rate-limiting enzyme of plant respiration and glycolysis metabolism, has been found to play a vital role in plant sugar sensing and sugar signal transduction. Using Jatropha curcas genome database and bioinformatics method, J. curcas HXK gene family (JcHXK) was identified and its phylogenetic evolution, functional domain, signal peptide at the N-terminal, and expression analysis were conducted. The results showed that a total of 4 HXK genes (JcHXK1, JcHXK2, JcHXK3, and JcHKL1) with 9 exons were systematically identified from J. curcas. JcHXK1, JcHXK3, and JcHKL1 with putative transmembrane domain at the N-terminal belonged to the type of secretory pathway protein, and JcHXK2 contained putative chloroplast targeting peptide. Quantitative real-time PCR (qRT-PCR) analysis revealed that all the four JcHXKs were expressed in different tissues of the leaves, roots, and seeds; however, JcHXK1 and JcHKL1 expression were higher in the roots, whereas JcHXK2 and JcHXK3 showed over-expression in the leaves and seeds, respectively. Furthermore, all the four JcHXKs were up-regulated in the leaves after cold stress at 12 °C; however, only JcHXK3 remarkably demonstrated cold-induced expression in the roots, which reached the highest expression level at 12 h (2.28-fold). According to the cis-acting element analysis results, JcHXK2 contained the most low temperature responsive elements, which was closely related to the cold resistance in J. curcas. A pET-28a-JcHXK2 prokaryotic recombinant expression vector was successfully constructed and a 57.0 kDa protein was obtained, JcHXK2 revealed catalytic activity towards glucose and fructose, with a higher affinity for glucose than fructose. The subcellular localization assays revealed that JcHXK2 was localized in the chloroplast. The results of this study might provide theoretical foundation for further studies on gene cloning and functional verification of HXK family in J. curcas.

Global Phosphoproteomic Analysis Reveals the Defense and Response Mechanisms of Jatropha Curcas Seedling under Chilling Stress.

As a promising energy plant for biodiesel, Jatropha curcas is a tropical and subtropical shrub and its growth is affected by one of major abiotic stress, chilling. Therefore, we adopt the phosphoproteomic analysis, physiological measurement and ultrastructure observation to illustrate the responsive mechanism of J. curcas seedling under chilling (4 °C) stress. After chilling for 6 h, 308 significantly changed phosphoproteins were detected. Prolonged the chilling treatment for 24 h, obvious physiological injury can be observed and a total of 332 phosphoproteins were examined to be significantly changed. After recovery (28 °C) for 24 h, 291 phosphoproteins were varied at the phosphorylation level. GO analysis showed that significantly changed phosphoproteins were mainly responsible for cellular protein modification process, transport, cellular component organization and signal transduction at the chilling and recovery periods. On the basis of protein-protein interaction network analysis, phosphorylation of several protein kinases, such as SnRK2, MEKK1, EDR1, CDPK, EIN2, EIN4, PI4K and 14-3-3 were possibly responsible for cross-talk between ABA, Ca2+, ethylene and phosphoinositide mediated signaling pathways. We also highlighted the phosphorylation of HOS1, APX and PIP2 might be associated with response to chilling stress in J. curcas seedling. These results will be valuable for further study from the molecular breeding perspective.

Biologically Active Orbitides from the Euphorbiaceae Family.

A comprehensive overview of natural orbitides isolated from Euphorbiaceae species and their most relevant biological activities are presented. Euphorbiaceae is a large and diverse family, which comprises about 300 genera, and is known as an important source of medicines and toxins. Several classes of secondary metabolites have been described for this taxon, however, orbitides have been broadly reported in Jatropha and Croton genera. Additionally, the latex is documented as the main source of orbitides in this family. Based on their structural and functional diversity, orbitides present a large variety of biological activities described as cytotoxicity, antimalarial, antibacterial, antifungal, enzymatic inhibition, and immunosuppressive, although the mechanism of action still needs to be further investigated. In recent years, the discovery of bioactive cyclic peptides from different sources has grown exponentially, making them promising molecules in the search for new drug leads. This review also highlights the attempts made by many researchers to organize the orbitides nomenclature and amino acid numbering, as well the important progress recently achieved in the biosynthetic study area.