Paper spray portable mass spectrometry for screening of phorbol ester contamination in glycerol-based medical products.

The Jatropha curcas plant (Jatropha) has been proposed as a source of biodiesel fuel, as it yields crude glycerol as an abundant by-product. Its by-products could serve as a starting material in making glycerol for FDA-regulated products. Jatropha is not regarded as a source of edible vegetable oil since it contains phorbol esters (PEs). PEs, even at very low exposure concentrations, demonstrate various toxicities in humans and animals, but may not be detected by routine impurity analyses. Here, we demonstrate the development of a rapid and simplified method for the detection and quantification of Jatropha-derived PE toxins using ambient ionization mass spectrometry. To do this, we successfully coupled a paper spray ambient ionization source with an ion trap portable mass spectrometer. The paper spray source was assembled using chromatography papers, and analyte ions were generated by applying a high voltage to a wetted paper triangle loaded with PE standards. For method development, we used commercially available PE standards on an ion trap portable mass spectrometer. Standard solutions were prepared using ethanol with PE concentrations ranging from 1.0 to 0.0001 mg mL-1. Spike and recovery experiments were performed using USP grade and commercially available glycerol. To discern chemical differences between samples, we applied multivariate data analysis. Based on the results obtained, paper spray coupled with a portable mass spectrometric method can be successfully adopted for the analysis of toxic contaminants present in glycerol-based consumer products with LOD and LOQ of 0.175 µg mL-1 and 0.3 µg mL-1 respectively. This direct, simple design, and low-cost sampling and ionization method enables fast screening with high sensitivity in non-laboratory settings.

Induction of Somatic Embryogenesis in Jatropha curcas.

Jatropha curcas has been a promising crop for biofuel production for the last decade. However, the lack of resistant materials to diseases and improved quality of the oil produced by the seeds has restricted the use of this promising crop. The genetic modifications in the fatty acid pathway, as well as the introduction of resistance to different diseases, would change the fate of Jatropha. To achieve these goals, we need to have a very efficient regeneration system. Here, we report a very useful protocol to induce somatic embryogenesis from leaves of Jatropha using cytokinin as the only growth regulator.

Deep proteome analysis of gerontoplasts from the inner integument of developing seeds of Jatropha curcas.

UNLABELLED: The inner integument of Jatropha curcas seeds is a non-photosynthetic tissue that acts primarily as a conduit for the delivery of nutrients to the embryo and endosperm. In this study we performed a histological and transmission electron microscopy analysis of the inner integument in stages prior to fertilization to 25days after pollination, to establish the structural changes associated with the plastid to gerontoplast transition. This study showed that plastids are subjected to progressive changes, which include the dismantling of the internal membrane system, matrix degradation and the formation of stromule-derived vesicles. A proteome analysis of gerontoplasts isolated from the inner integument at 25days after pollination, resulted in the identification of 1923 proteins, which were involved in a myriad of metabolic functions, such as synthesis of amino acids and fatty acids. Among the identified proteins, were also a number of hydrolases (peptidases, lipases and carbohydrases), which presumably are involved in the ordered dismantling of this organelle to provide additional sources of nutrients for the growing embryo and endosperm. The dataset we provide here may provide a foundation for the study of the proteome changes associated with the plastid to gerontoplast transition in non-photosynthetic tissues. SIGNIFICANCE: We describe ultrastructural features of gerontoplasts isolated from the inner integument of developing seeds of Jatropha curcas, together with a deep proteome analysis of these gerontoplasts. This article explores a new aspect of the biology of plastids, namely the ultrastructural and proteome changes associated with the transition plastid to gerontoplast in a non-photosynthetic tissue.

Proteomic Analysis of the Endosperm Ontogeny of Jatropha curcas L. Seeds.

Seeds of Jatropha curcas L. represent a potential source of raw material for the production of biodiesel. However, this use is hampered by the lack of basic information on the biosynthetic pathways associated with synthesis of toxic diterpenes, fatty acids, and triacylglycerols, as well as the pattern of deposition of storage proteins during seed development. In this study, we performed an in-depth proteome analysis of the endosperm isolated from five developmental stages which resulted in the identification of 1517, 1256, 1033, 752, and 307 proteins, respectively, summing up 1760 different proteins. Proteins with similar label free quantitation expression pattern were grouped into five clusters. The biological significance of these identifications is discussed with special focus on the analysis of seed storage proteins, proteins involved in the metabolism of fatty acids, carbohydrates, toxic components and proteolytic processing. Although several enzymes belonging to the biosynthesis of diterpenoid precursors were identified, we were unable to find any terpene synthase/cyclase, indicating that the synthesis of phorbol esters, the main toxic diterpenes, does not occur in seeds. The strategy used enabled us to provide a first in depth proteome analysis of the developing endosperm of this biodiesel plant, providing an important glimpse into the enzymatic machinery devoted to the production of C and N sources to sustain seed development.

Effect of plant growth regulators on fatty acids composition in Jatropha curcas L. callus culture.

The influence of Naphtaleneacetic acid (NAA) and 6-Benzylaminopurine (BAP) on callus formation, its morphology and fatty acids profile were examined from Jatropha curcas L. Embryo from seeds of J. curcas L. were sown in Murashige and skoog (MS) medium with NAA and BAP. All treatments induced callus formation, however callus morphology was different in most of the treatments. Higher callus biomass was presented with 1.0 NAA + 0.5 BAP mg/L. Plant growth regulators modifies the fatty acids profile in callus of J. curcas L. BAP was induced linoleic and linolenic acids.

Proteome analysis of the inner integument from developing Jatropha curcas L. seeds.

In this study, we performed a systematic proteomic analysis of the inner integument from developing seeds of Jatropha curcas and further explored the protein machinery responsible for generating the carbon and nitrogen sources to feed the growing embryo and endosperm. The inner integument of developing seeds was dissected into two sections called distal and proximal, and proteins were extracted from these sections and from the whole integument and analyzed using an EASY-nanoLC system coupled to an ESI-LTQ-Orbitrap Velos mass spectrometer. We identified 1526, 1192, and 1062 proteins from the proximal, distal, and whole inner integuments, respectively. The identifications include those of peptidases and other hydrolytic enzymes that play a key role in developmental programmed cell death and proteins associated with the cell-wall architecture and modification. Because many of these proteins are differentially expressed within the integument cell layers, these findings suggest that the cells mobilize an array of hydrolases to produce carbon and nitrogen sources from proteins, carbohydrates, and lipids available within the cells. Not least, the identification of several classes of seed storage proteins in the inner integument provides additional evidence of the role of the seed coat as a transient source of reserves for the growing embryo and endosperm.

Bioactivities of Jc-SCRIP, a type 1 ribosome-inactivating protein from Jatropha curcas seed coat.

In this study, a type 1 RIP, designated as Jc-SCRIP, was first isolated from the seed coat of Jatropha curcas Linn. It was purified by ammonium sulfate precipitation and chromatography on DEAE-Sephacel™ and CM-cellulose columns. Purification fold of Jc-SCRIP increased 113.8 times, and the yield was 1.13% of the total protein in the final step. It was shown to be a monomeric glycoprotein with a molecular mass of 38 938 Da, as determined by MALDI-TOF/MS. It exhibited hemagglutination activity and possessed strong N-glycosidase activity. The antimicrobial activity of Jc-SCRIP was tested against nine human pathogenic bacteria and one fungus; the most potent inhibitory activity was against Staphylococcus epidermidis ATCC 12228, with minimum inhibitory concentration value of 0.20 µm. Jc-SCRIP demonstrated in vitro cytotoxicity against human breast adenocarcinoma cell line (MCF-7), a colon adenocarcinoma (SW620), and a liver carcinoma cell line (HepG2), with IC50 values of 0.15, 0.25, and 0.40 mm, respectively. The results suggested that Jc-SCRIP may be a potential natural antimicrobial and anticancer agent in medical applications.

Optimization of supercritical methanol reactive extraction by response surface methodology and product characterization from Jatropha curcas L. seeds.

In this study, optimization of supercritical reactive extraction directly from Jatropha seeds in a high pressure batch reactor using Response Surface Methodology (RSM) coupled with Central Composite Rotatable Design (CCRD) was performed. Four primary variables (methanol to solid ratio (SSR), reaction temperature, time and CO2 initial pressure) were investigated under the proposed constraints. It was found that all variables had significant effects towards fatty acid methyl esters (FAME) yield. Moreover, three interaction effects between the variables also played a major role in influencing the final FAME yield. Optimum FAME yield at 92.0 wt.% was achieved under the following conditions: 5.9 SSR, 300°C, 12.3 min and 20 bar CO2. Final FAME product was discovered to fulfil existing international standard. Preliminary characterization analysis proved that the solid residue can be burnt as solid fuel in the form of biochar while the liquid product can be separated as specialty chemicals or burned as bio-oil for energy production.

A method for seedling recovery in Jatropha curcas after cryogenic exposure of the seeds.

Actually, the germplasm of Jatropha spp. is conserved as whole plants in field collections. Under this storage method, the genetic resources are exposed to disease, pest and natural hazards such as human error, drought and weather damage. Besides, field genebanks are costly to maintain and with important requirements of trained personnel. Thus, the development of efficient techniques to ensure its safe conservation and regeneration is therefore of paramount importance. In this work we describe a method for Jatropha curcas seeds cryoexposure and seedling recovery after thawed. In a first experiment, an efficient protocol for in vitro plant recovery was carried out using zygotic embryo or seeds with or without coat. In a second experiment, desiccated seeds with or without coat were exposed to liquid nitrogen and evaluated after cryoexposure. Germination percentages were variable among treatments, and seeds demonstrated tolerance to liquid nitrogen exposure under certain conditions. Seeds of J. curcas presented up to 99.6% germination after seed coat removal. Seeds with coat cultured in vitro did not germinate, and were 60% contaminated. The germination of the zygotic embryos was significantly higher in the 1/2 MS medium (93.1%) than in WPM medium (76.2%), but from zygotic embryo, abnormal seedlings reached up to 99%. Seeds with coat exposed to liquid nitrogen showed 60% germination in culture after coat removal with good plant growth, and seeds cryopreserved without coat presented 82% germination, but seedlings showed a reduced vigor and a significant increase in abnormal plants. Seeds cultured in vitro with coat did not germinate, independently of cryoexposure or not. This study reports the first successful in vitro seedling recovery methodology for Jatropha curcas seeds, after a cryopreservation treatment, and is recommended as an efficient procedure for in vitro plant recovery, when seeds are conserved in germplasm banks by low or cryotemperatures.

A method for seedling recovery in Jatropha curcas after cryogenic exposure of the seeds

Actually, the germplasm of Jatropha spp. is conserved as whole plants in field collections. Under this storage method, the genetic resources are exposed to disease, pest and natural hazards such as human error, drought and weather damage. Besides, field genebanks are costly to maintain and with important requirements of trained personnel. Thus, the development of efficient techniques to ensure its safe conservation and regeneration is therefore of paramount importance. In this work we describe a method for Jatropha curcas seeds cryoexposure and seedling recovery after thawed. In a first experiment, an efficient protocol for in vitro plant recovery was carried out using zygotic embryo or seeds with or without coat. In a second experiment, desiccated seeds with or without coat were exposed to liquid nitrogen and evaluated after cryoexposure. Germination percentages were variable among treatments, and seeds demonstrated tolerance to liquid nitrogen exposure under certain conditions. Seeds of J. curcas presented up to 99.6% germination after seed coat removal. Seeds with coat cultured in vitro did not germinate, and were 60% contaminated. The germination of the zygotic embryos was significantly higher in the ½ MS medium (93.1%) than in WPM medium (76.2%), but from zygotic embryo, abnormal seedlings reached up to 99%. Seeds with coat exposed to liquid nitrogen showed 60% germination in culture after coat removal with good plant growth, and seeds cryopreserved without coat presented 82% germination, but seedlings showed a reduced vigor and a significant increase in abnormal plants. Seeds cultured in vitro with coat did not germinate, independently of cryoexposure or not. This study reports the first successful in vitro seedling recovery methodology for Jatropha curcas seeds, after a cryopreservation treatment, and is recommended as an efficient procedure for in vitro plant recovery, when seeds are conserved in germplasm banks by low or cryotemperatures.

Actualmente, el germoplasma de las especies de Jatropha ssp. se conserva como plantas enteras en las colecciones de campo. Bajo este método de almacenamiento, los recursos genéticos están expuestos a enfermedades, plagas y desastres naturales tales como el error humano, la sequía y las inclemencias del tiempo. Además, los bancos de germoplasma de campo son costosos de mantener y requieren bastante personal capacitado. Por lo tanto, el desarrollo de técnicas eficientes para asegurar su conservación segura así como su regeneración, es de suma importancia. En este trabajo se describe un método de recuperación para semillas y plántulas crioexpuestas de Jatropha curcas después de descongeladas. En un primer experimento, se llevó a cabo un protocolo eficiente para la recuperación de plantas in vitro mediante el uso de embriones cigóticos o semillas con o sin testa. En un segundo experimento, las semillas disecadas, con o sin testa fueron expuestas a nitrógeno líquido y se evaluaron después de la crioexposición. Los porcentajes de germinación fueron variables entre los tratamientos, y las semillas demostraron tolerancia a la exposición del nitrógeno líquido bajo ciertas condiciones. Las semillas de J. curcas presentaron hasta un 99.6% de germinación después de la eliminación de la testa. Las semillas con la testa cultivadas in vitro no germinaron, y el 60% se contaminaron. La germinación de los embriones cigóticos fue significativamente alta en el medio ½ MS (93.1%) en comparación con el medio WPM (76.2%), pero desde los embriones zigóticos, las plántulas anormales alcanzaron más del 99%. Semillas con la testa inmersa en nitrógeno líquido mostraron un 60% de germinacion en cultivos despúes de la remoción de la testa con un buen crecimiento de la planta, y las semillas criopreservadas sin testa presentaron un 82% de germinación, pero las plántulas mostraron un reducido vigor y un incremento significativo de plantas anormales. Semillas con testa cultivadas in vitro no germinaron, independientemente de la criopreservación o no. Este estudio reporta el primer éxito in vitro de una metodología de recuperación de plántulas para semillas de Jatropha curcas, después de un tratamiento de criopreservación, que se recomienda como un procedimiento eficaz para la recuperación de plantas in vitro, cuando las semillas se conservan en bancos de germoplasma a bajas o crio-temperaturas.

Regeneration of Jatropha curcas through efficient somatic embryogenesis and suspension culture.

.Using immature zygotic embryos as explants, we have developed an efficient method for somatic embryogenesis in three germplasm accessions collected from China, India and Indonesia. Indirect somatic embryogenesis was achieved when endosperm tissue and immature embryos between 0.5-1.0 cm in length were cultured in a medium with 2,4-D, preferably at 5-10 mg/l, followed by a shift to a hormone-free medium supplemented with glutamine and asparagine. Production of secondary embryos was improved by supplementing KNO3, glutamine and asparagine. 2,4-D (0.1-0.2 mg/l). PEG 8000 (5-10%) were essential for maintenance of embryogenic calli in liquid medium. Regeneration of soil-ready plants took as short as 3 months using the suspension cultures. Over 95% of the regenerated trees were able to flower and set seeds with no discernable morphological abnormality. This regeneration method is expected to facilitate the development of more efficient transformation system for Jatropha curcas.

Reactive extraction of Jatropha curcas L. seed for production of biodiesel: process optimization study.

Biodiesel from Jatropha curcas L. seed is conventionally produced via a two-step method: extraction of oil and subsequent esterification/transesterification to fatty acid methyl esters (FAME), commonly known as biodiesel. Contrarily, in this study, a single step in situ extraction, esterification and transesterification (collectively known as reactive extraction) of J. curcas L. seed to biodiesel, was investigated and optimized. Design of experiments (DOE) was used to study the effect of various process parameters on the yield of FAME. The process parameters studied include reaction temperature (30-60 degrees C), methanol to seed ratio (5-20 mL/g), catalyst loading (5-30 wt %), and reaction time (1-24 h). The optimum reaction condition was then obtained by using response surface methodology (RSM) coupled with central composite design (CCD). Results showed that an optimum biodiesel yield of 98.1% can be obtained under the following reaction conditions: reaction temperature of 60 degrees C, methanol to seed ratio of 10.5 mL/g, 21.8 wt % of H(2)SO(4), and reaction period of 10 h.

Fermentable sugar release from Jatropha seed cakes following lime pretreatment and enzymatic hydrolysis.

Composition change of Jatropha seed cake samples was evaluated upon lime pretreatment at 100 degrees C with different parameters. With a lime dose of 0.2 g and a water content of 10 ml per gram of cake and a treatment period of 1 h, 38.2+/-0.6% of lignin was removed. However, 65+/-16% of hemicellulose was also lost under this condition. For all the treatments tested, cellulose content was not affected by lime supplementation. Through further examining total reducing sugar (TRS) release by enzymatic hydrolysis after lime pretreatment, we have found that 0.1 g of lime and 9 ml of water per gram of cake and 3 h pretreatment produced the maximal 68.9% conversion of cellulose. Without lime pretreatment, the highest cellulose conversion was 33.3%. One microalgal species, Schizochytrium limacinum SR21 was able to grow on the hydrolyzates and generate a biomass density of 3.2 g/l in 4 days.

Novel 12-deoxy-16-hydroxyphorbol diesters isolated from the seed oil of Jatropha curcas.

Six unstable intramolecular diterpene esters were isolated from the seed oil of Jatropha curcas. Five of these, Jatropha factors C(2)-C(6) (3-7), are new natural products, and the structure of the known Jatropha factor C(1) (2) has been revised. All compounds possess the same diterpene moiety, namely, 12-deoxy-16-hydroxyphorbol (1). The dicarboxylic acid moieties of 2-5 contain a bicyclo[3.1.0]hexane unit, and those of 6 and 7 a cyclobutane unit, which is described for the first time within this compound class. Compounds 4 and 5 are C-8' epimers. The structures of 2-7 were elucidated by spectroscopic methods and give an insight into the biogenesis of the characterized substances.