Seed characteristics and fatty acid composition of castor (<i>Ricinus communis</i> L.) varieties in Northeast China

Oil content and fatty acid composition were investigated on 12 castor varieties and strains by using the soxhlet extraction method and capillary gas chromatography. This was made to provide a reference and theoretical basis for castorbean breeding with high oil content, determine variability of seed compounds for breeding purposes, and broaden chemical material choices. Results revealed that crude fat percentage in seeds ranged from 18.91 to 35.84% with an average of 25.91%; the absolute content of ricinoleic acid varied between 171.65 g/kg and 314.03 g/kg with an average of 222.43 g/kg, and kernel crude fat percentage was between 24.28 and 46.97% with an average of 34.30%. All these study variables were highest in the 2129 strain. The percentage of ricinoleic acid in crude fat was between 83.85 to 87.62%, and the highest value was found in the zhebi4 accession. The other fatty acids appeared in small concentrations, and showed small amplitude: 1.12 to 1.61%, 1.21 to 1.61%, 3.53 to 4.80%, 5.35 to 6.38%, 0.52 to 0.79%, 0.05 to 0.08% and 0.43 to 0.55%, for palmitic, stearic, oleic, linolic, linolenic, arachidic, and arachidonic acids, respectively. One hundred seed weight was determined for each accession. One hundred seed weight ranged from 25.7 g to 34.0 g with an average of 29.9 g. There was a significant correlation between seed weight and oil content, but the correlation value was low (r=0.51). Cluster analysis by SSPS based on the content of fatty acid composition revealed that the accessions were divided into three independent clusters. These findings will clearly provide useful information for further research in breeding and utilization of castor oil.     

Proteomic Analyses of Three Inflorescence Styles of Castor (<i>Ricinus communis</i> L.) at Different Developmental Stages

Castor (Ricinus communis L.) is one of ten oil crops in the world and has complex inflorescence styles. Generally, castor has three inflorescence types: single female inflorescence (SiFF), standard female inflorescence (StFF) and bisexual inflorescence (BF). StFF is realized as a restorer line and as a maintainer line, which was applied to castor hybrid breeding. However, the developmental mechanism of the three inflorescences is not clear. Therefore, we used proteomic techniques to analyze different inflorescence styles. A total of 72 diferentially abundant protein species (DAPs) were detected. These DAPs are primarily involved in carbon and energy metabolism and carbon fixation in the photosynthetic organism pathway. The results showed that DAPs are involved in photosynthesis to control the distribution of imported carbohydrates and exported photoassimilates and thus affect the inflorescence development of castor. In addition, these DAPs are also involved in cysteine and methionine metabolism. Quantitative real-time PCR (qRT-PCR) results demonstrated that the proteomics data collected in this study were reliable. Our findings indicate that the carbon cycle and amino acid metabolism influence the inflorescence development of castor.     

Investigation of <i>GhFAT</i> Genes Related to Seed Oil Content and Fatty Acid Composition in <i>Gossypium hirsutum</i> L.

Fatty Acyl-ACP thioesterase (FAT) is a key enzyme controlling oil biosynthesis in plant seeds. FATs can be divided into two subfamilies, FATA and FATB according to their amino acid sequences and substrate specificity. The Upland cotton genome contains 20 GhFAT genes, amongst which 6 genes were of the GhFATA subfamily and 14 of the GhFATB subfamily. The 20 GhFAT genes are unevenly distributed on 14 chromosomes. The GhFATA genes have 5 or 7 exons and the GhFATB genes have 6 or 7 exons. All GhFAT proteins have the conserved Acyl-ACP_TE domain and PLN02370 super family, the typical characteristics of plant thioesterases. Analyses of the expression level of GhFATs and the compositions of fatty acid in 5–60 days-post-anthesis seeds showed that the ratio of saturated fatty acids to unsaturated fatty acids was consistent with the expression profile of GhFATB12, GhFATB3, and GhFATB10; the ratio of monounsaturated fatty acid to polyunsaturated fatty acids was consistent with the expression profile of GhFATA3. The oil contents of mature cottonseeds were positively correlated with the contents of palmitic acid and linolenic acid as well as seed vigor. These results provide essential information for further exploring the role(s) of the specific GhFATs in determining oil biosynthesis and cottonseed compositions.     

Acyl-ACP thioesterases from castor (Ricinus communis L.): An enzymatic system appropriate for high rates of oil synthesis and accumulation

Acyl–acyl carrier protein (ACP) thioesterases are enzymes that terminate the intraplastidial fatty acid synthesis in plants by hydrolyzing the acyl-ACP intermediates and releasing free fatty acids to be incorporated into glycerolipids. These enzymes are classified in two families, FatA and FatB, which differ in amino acid sequence and substrate specificity. In the present work, both FatA and FatB thioesterases were cloned, sequenced and characterized from castor (Ricinus communis) seeds, a crop of high interest in oleochemistry. Single copies of FatA and FatB were found in castor resulting to be closely related with those of Jatropha curcas. The corresponding mature proteins were heterologously expressed in Escherichia coli for biochemical characterization after purification, resulting in high catalytic efficiency of RcFatA on oleoyl-ACP and palmitoleoyl-ACP and high efficiencies of RcFatB for oleoyl-ACP and palmitoyl-ACP. The expression profile of these genes displayed the highest levels in expanding tissues that typically are very active in lipid biosynthesis such as developing seed endosperm and young expanding leaves. The contribution of these two enzymes to the synthesis of castor oil is discussed.     

Comparative proteomics of seed maturation in oilseeds reveals differences in intermediary metabolism

Proteomics is increasingly being used to understand enzyme expression and regulatory mechanisms involved in the accumulation of storage reserves in crops with sequenced genomes. During the past six years, considerable progress has been made to characterize proteomes of both mature and developing seeds, particularly oilseeds - plants which accumulate principally oil and protein as storage reserves. This review summarizes the emerging proteomics data, with emphasis on seed filling in soy, rapeseed, castor and Arabidopsis as each of these oilseeds were analyzed using very similar proteomic strategies. These parallel studies provide a comprehensive view of source-sink relationships, specifically sucrose assimilation into organic acid intermediates for de novo amino acid and fatty acid synthesis. We map these biochemical processes for seed maturation and illustrate the differences and similarities among the four oilseeds. For example, while the four oilseeds appear capable of producing cytosolic phosphoenolpyruvate as the principal carbon intermediate, soybean and castor also express malic enzymes and malate dehydrogenase, together capable of producing malate that has been previously proposed to be the major intermediate for fatty acid synthesis in castor. We discuss these and other differences in the context of intermediary metabolism for developing oilseeds.     

Sunflower (Helianthus annuus) long‐chain acyl‐coenzyme A synthetases expressed at high levels in developing seeds

Long chain fatty acid synthetases (LACSs) activate the fatty acid chains produced by plastidial de novo biosynthesis to generate acyl‐CoA derivatives, important intermediates in lipid metabolism. Oilseeds, like sunflower, accumulate high levels of triacylglycerols (TAGs) in their seeds to nourish the embryo during germination. This requires that sunflower seed endosperm supports very active glycerolipid synthesis during development. Sunflower seed plastids produce large amounts of fatty acids, which must be activated through the action of LACSs, in order to be incorporated into TAGs. We cloned two different LACS genes from developing sunflower endosperm, HaLACS1 and HaLACS2, which displayed sequence homology with Arabidopsis LACS9 and LACS8 genes, respectively. These genes were expressed at high levels in developing seeds and exhibited distinct subcellular distributions. We generated constructs in which these proteins were fused to green fluorescent protein and performed transient expression experiments in tobacco cells. The HaLACS1 protein associated with the external envelope of tobacco chloroplasts, whereas HaLACS2 was strongly bound to the endoplasmic reticulum. Finally, both proteins were overexpressed in Escherichia coli and recovered as active enzymes in the bacterial membranes. Both enzymes displayed similar substrate specificities, with a very high preference for oleic acid and weaker activity toward stearic acid. On the basis of our findings, we discuss the role of these enzymes in sunflower oil synthesis.     

转录组测序揭示油桐脂肪酸的合成途径

油桐是我国重要的木本油料植物,过去对油桐的研究主要集中于栽培和常规育种,与油桐种仁油脂合成相关的分子机理研究还未见报道。本研究采用转录组测序（RNA-Seq）技术,比较了油桐种子油脂合成3个不同时期的转录组,获得了大量差异表达的Unigene序列。通过GO（GeneOntology）分类和代谢途径富集性分析将这些差异表达的Unigene归类于包含脂肪酸生物合成途径在内的128个代谢途径。随后将脂肪酸生物合成途径中的54个Unigene序列在KEGG（KyotoEncyclopediaofGenesandGenomes）数据库中进行比对,获得了14个关键酶的同源蛋白质。本研究通过对编码这些同源蛋白质的基因在油桐种子油脂合成期的表达模式进行分析,以期为油桐油脂合成,尤其是桐酸合成机理的解析提供理论参考,并为进一步的理论研究和油桐的遗传改良提供潜在的基因资源,从而提高油桐的单位面积产量。     

基于油桐种子3个不同发育时期转录组的油脂合成代谢途径分析

油桐是我国重要的木本油料植物, 过去对油桐的研究主要集中于栽培和常规育种, 与油桐种仁油脂合成相关的分子机理研究还未见报道。文章采用RNA-seq技术对油桐种子油脂合成的3个不同时期的转录组进行比较, 获得了大量差异表达的Unigene序列。在此基础上, 通过GO分类和Pathway富集性分析将这些差异表达Unigene归类于128个代谢途径, 其中包含与油脂合成相关的脂肪酸生物合成和甘油磷脂代谢途径。桐酸经脂肪酸生物合成途径合成后通过甘油磷脂代谢途径以桐油的形式贮存。将这两个代谢途径的Unigene序列在KEGG数据库中进行比对, 获得了一些关键酶的同源蛋白质。文章通过对编码这些同源蛋白质的基因在油桐种子油脂合成期的表达模式进行分析, 以期为油桐油脂合成, 尤其是桐酸合成机理的解析提供理论参考, 并为油桐的遗传改良提供潜在的基因资源, 从而提高油桐的单位面积产量。     

Bioinformatics and Functional Analysis of High Oleic Acid-Related Gene Gm<i>SAM22</i> in Soybean [<i>Glycine max</i> (L.) Merr.]

High yield, high quality, stable yield, adaptability to growth period, and modern mechanization are the basic requirements for crops in the 21st century. Soybean oleic acid is a natural unsaturated fatty acid with strong antioxidant properties and stability. Known as a safe fatty acid, it has the ability to successfully prevent cardiovascular and cerebrovascular disorders. Improving the fatty acid composition of soybean seeds, can not only speed up the breeding process of high-quality high-oil and high-oleic soybeans, but also have important significance in human health, and provide the possibility for the development of soybean oil as a new energy source. Hence, the aim of this study was to analyze the high oleic acid elated gene GmSAM22 in soybean. In this research the soybean oleic acid-related gene GmSAM22 was screened out by Genome-wide association analysis, a 662 bp fragment was acquired by specific PCR amplification, and the pMD18T cloning vector was linked by the use of a seamless cloning technique. Bioinformatics analysis of the signal peptide prediction, subcellular localization, protein hydrophobicity, transmembrane region analysis, a phosphorylation site, protein secondary and tertiary structure and protein interaction analysis of the protein encoded by the SAM22 gene was carried out. The plasmid of the gene editing vector is pBK041. The overexpression vector was transformed from pCAMBIA3301 as the base vector, and overexpression vector were designed. Positive plants were obtained by genetic transformation by the pollen tube channel method. Fluorescence quantitative PCR was performed on the T₂ generation plants to detect the relative expression levels in different tissues. Southern Blot was used to detect the presence of hybridization signal. Screening genes BAR, 35S, and NOS in plants were identified by conventional PCR. 10 seeds with high and low oleic acid content were chosen for quantitative PCR identification, and finally, the concentration and morphology of soybean fatty acids were identified by near-far infrared spectroscopy. On 10 seeds with an upper and lower oleic acid content, a quantitative fluorescence analysis was done. In Southern blot hybridization, the SAM22 gene was integrated into the recipient soybean plant in hands of a sole copy. Fluorescence quantitative PCR appeared that the average relative expression of the SAM22 gene in roots, stems, leaves, and seeds was 1.70, 1.67, 3.83, and 4.41, respectively. Positive expression seeds had a 4.77% increase in oleic acid content. The level of oleic acid in the altered seeds was reduced by 4.13% when compared to CK, and it was discovered that the GmSAM22 gene could be a regulatory and secondary gene that promotes the conversion of stearic acid to oleic acid in soybean. There has not been a discussion of gene cloning or functional verification. The cloning and genetic transformation of the soybean SAM22 gene can effectively increase the content of oleic acid, which lays a foundation for the study of soybean with high oleic acid.     

Cytosolic Phosphorylating Glyceraldehyde-3-Phosphate Dehydrogenases Affect Arabidopsis Cellular Metabolism and Promote Seed Oil Accumulation

The cytosolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPC) catalyzes a key reaction in glycolysis, but its contribution to plant metabolism and growth are not well defined. Here, we show that two cytosolic GAPCs play important roles in cellular metabolism and seed oil accumulation. Knockout or overexpression of GAPCs caused significant changes in the level of intermediates in the glycolytic pathway and the ratios of ATP/ADP and NAD(P)H/NAD(P). Two double knockout seeds had ∼3% of dry weight decrease in oil content compared with that of the wild type. In transgenic seeds under the constitutive 35S promoter, oil content was increased up to 42% of dry weight compared with 36% in the wild type and the fatty acid composition was altered; however, these transgenic lines exhibited decreased fertility. Seed-specific overexpression lines had >3% increase in seed oil without compromised seed yield or fecundity. The results demonstrate that GAPC levels play important roles in the overall cellular production of reductants, energy, and carbohydrate metabolites and that GAPC levels are directly correlated with seed oil accumulation. Changes in cellular metabolites and cofactor levels highlight the complexity and tolerance of Arabidopsis thaliana cells to the metabolic perturbation. Further implications for metabolic engineering of seed oil production are discussed.     

Allantoin Alleviates Seed Germination Thermoinhibition in <i>Arabidopsis</i>

Allantoin as the metabolite of purine catabolism can store and remobilize nitrogen for plant growth and development. However, emerging evidence suggests it also contributes to plant tolerance to stress response through altering abscisic acid (ABA) and reducing reactive oxygen species (ROS) level. 1-CYS PEROXIREDOXIN (PER1) is a seed-specific antioxidant that enhances seed longevity through scavenging ROS over-accumulation. High temperature (HT) suppresses seed germination and induces seed secondary dormancy, called as seed germination thermoinhibition. However, the mechanism that allantoin and PER1 regulate seed germination thermoinhibition remains unknown. In this study, we reported that allantoin treatment enhances seed germination under HT stress. Consistently, the aln mutants displayed higher seed germination, as well as more accumulation of endogenous allantoin, than that of wild-type control. Further biochemical and genetic analyses showed that allantoin reduces ABA content under HT, and allantoin targets PER1 to efficiently scavenge HT-induced ROS accumulation, meanwhile, the function of allantoin requires PER1 during seed gemination thermotolerance. Collectively, our finding proposes a novel function of allantoin in enhancing seed germination tolerance to HT, and uncovers the underlying mechanism by which allantoin regulates seed germination through altering ABA metabolism and PER1-mediated ROS level under HT stress.     

Components of complex lipid biosynthetic pathways in developing castor (Ricinus communis) seeds identified by MudPIT analysis of enriched endoplasmic reticulum

Ricinoleic acid is a feedstock for nylon-11 (N11) synthesis which is currently obtained from castor (Ricinus communis) oil. Production of this fatty acid in a temperate oilseed crop is of great commercial interest, but the highest reported level in transgenic plant oils is 30%, below the 90% observed in castor and insufficient for commercial exploitation. To identify castor oil-biosynthetic enzymes and inform strategies to improve ricinoleic acid yields, we performed MudPIT analysis on endoplasmic reticulum (ER) purified from developing castor bean endosperm. Candidate enzymes for all steps of triacylglycerol synthesis were identified among 72 proteins in the data set related to complex-lipid metabolism. Previous reported proteomic data from oilseeds had not included any membrane-bound enzyme that might incorporate ricinoleic acid into oil. Analysis of enriched ER enabled determination of which protein isoforms for these enzymes were in developing castor seed. To complement this data, quantitative RT-PCR experiments with castor seed and leaf RNA were performed for orthologues of Arabidopsis oil-synthetic enzymes, determining which were highly expressed in the seed. These data provide important information for further manipulation of ricinoleic acid content in oilseeds and peptide data for future quantification strategies.     

Effects of Heat Stress during Seed Filling Stage on <i>Brassica napus</i> Seed Oil Accumulation and Chlorophyll Fluorescence Characteristics

As global temperature rise, the threat of heat stress to rapeseed production is becoming more obvious. Exploring the response characteristics of two important biological pathways, oil accumulation and photosynthesis, to heat stress during B. napus seed filling is helpful in the genetic improvement of heat-tolerant rapeseed. The effects of heat stress on seed oil accumulation and chlorophyll fluorescence characteristics of 29 B. napus germplasms with different oil content and environmental sensitivity, including 6 rapeseed varieties which exhibited environment-sensitive/insensitive and with high, medium or low oil content, were tested by whole plant heat stress or the in vitro silique culture system. Both assay exhibited similar trend on oil content of the rapeseed germplasms. The heat effect on the chlorophyll fluorescence kinetic parameters F_v/F_m, ETR and Y(II) were also consistent. Heat stress significantly decreased oil content, although there was abundant genetic variation on heat tolerance among the genotypes. Correlation analysis showed that the decrease rate of F_v/F_m of silique heat-stressed B. napus developing seed was positive correlative to the decrease rate of mature seed oil content of the whole plant heat-stressed rapeseed (R = 0.9214, P-value < 0.01). Overall, the results indicated that heat stress inhibited oil accumulation and photosynthesis in B. napus developing seed. The decrease rate of chlorophyll fluorescence parameter F_v/F_m of heat-stressed developing seed could be used as the index of heat tolerant rapeseed identification. Further, two heat insensitive rapeseed varieties with high oil content were identified.     

Contrapuntal networks of gene expression during Arabidopsis seed filling

We have used cDNA microarrays to examine changes in gene expression during Arabidopsis seed development and to compare wild-type and mutant wrinkled1 (wri1) seeds that have an 80% reduction in oil. Between 5 and 13 days after flowering, a period preceding and including the major accumulation of storage oils and proteins, approximately 35% of the genes represented on the array changed at least twofold, but a larger fraction (65%) showed little or no change in expression. Genes whose expression changed most tended to be expressed more in seeds than in other tissues. Genes related to the biosynthesis of storage components showed several distinct temporal expression patterns. For example, a number of genes encoding core fatty acid synthesis enzymes displayed a bell-shaped pattern of expression between 5 and 13 days after flowering. By contrast, the expression of storage proteins, oleosins, and other known abscisic acid-regulated genes increased later and remained high. Genes for photosynthetic proteins followed a pattern very similar to that of fatty acid synthesis proteins, implicating a role in CO(2) refixation and the supply of cofactors for oil synthesis. Expression profiles of key carbon transporters and glycolytic enzymes reflected shifts in flux from cytosolic to plastid metabolism. Despite major changes in metabolism between wri1 and wild-type seeds, <1% of genes differed by more than twofold, and most of these were involved in central lipid and carbohydrate metabolism. Thus, these data define in part the downstream responses to disruption of the WRI1 gene.     

葵花籽油的酶辅助压榨制备工艺优化

葵花籽是一种优质的油料资源,是世界第四大油料作物。葵花籽不但含油量高,葵花籽油以其高达90%的不饱和脂肪酸和富含维生素E、胡萝卜素等营养成分的特点,被称为"保健油",有延缓衰老、调节新陈代谢和降低胆固醇等功能。采用酶辅助压榨法制备葵花籽油,在提高出油率的同时保留油脂的天然风味。通过单因素实验法研究了酶的种类、酶添加量、pH值、液固比、温度和时间对压榨葵花籽油出油率的影响,以确定最佳的工艺条件。结果表明,最佳工艺条件为：选用纤维素酶,酶添加量为0.7%,pH为4.5,液固比为25%,酶解温度为55℃,酶解时间为2.5 h。在此最佳条件下,出油率是传统冷榨法的3.48倍,油脂提取率达85%。     

Dynamic Metabolic Profiles and Tissue-Specific Source Effects on the Metabolome of Developing Seeds of Brassica napus

Canola (Brassica napus) is one of several important oil-producing crops, and the physiological processes, enzymes, and genes involved in oil synthesis in canola seeds have been well characterized. However, relatively little is known about the dynamic metabolic changes that occur during oil accumulation in seeds, as well as the mechanistic origins of metabolic changes. To explore the metabolic changes that occur during oil accumulation, we isolated metabolites from both seed and silique wall and identified and characterized them by using gas chromatography coupled with mass spectrometry (GC-MS). The results showed that a total of 443 metabolites were identified from four developmental stages. Dozens of these metabolites were differentially expressed during seed ripening, including 20 known to be involved in seed development. To investigate the contribution of tissue-specific carbon sources to the biosynthesis of these metabolites, we examined the metabolic changes of silique walls and seeds under three treatments: leaf-detachment (Ld), phloem-peeling (Pe), and selective silique darkening (Sd). Our study demonstrated that the oil content was independent of leaf photosynthesis and phloem transport during oil accumulation, but required the metabolic influx from the silique wall. Notably, Sd treatment resulted in seed senescence, which eventually led to a severe reduction of the oil content. Sd treatment also caused a significant accumulation of fatty acids (FA), organic acids and amino acids. Furthermore, an unexpected accumulation of sugar derivatives and organic acid was observed in the Pe- and Sd-treated seeds. Consistent with this, the expression of a subset of genes involved in FA metabolism, sugar and oil storage was significantly altered in Pe and Sd treated seeds. Taken together, our studies suggest the metabolite profiles of canola seeds dynamically varied during the course of oil accumulation, which may provide a new insight into the mechanisms of the oil accumulation at the metabolite level.     

A fatty acid condensing enzyme from Physaria fendleri increases hydroxy fatty acid accumulation in transgenic oilseeds of Camelina sativa

Main conclusion

Co-expression of a lesquerella fatty acid elongase and the castor fatty acid hydroxylase in camelina results in higher hydroxy fatty acid containing seeds with normal oil content and viability. Producing hydroxy fatty acids (HFA) in oilseed crops has been a long-standing goal to replace castor oil as a renewable source for numerous industrial applications. A fatty acid hydroxylase, RcFAH, from Ricinus communis, was introduced into Camelina sativa, but yielded only 15 % of HFA in its seed oil, much lower than the 90 % found in castor bean. Furthermore, the transgenic seeds contained decreased oil content and the germination ability was severely affected. Interestingly, HFA accumulation was significantly increased in camelina seed when co-expressing RcFAH with a fatty acid condensing enzyme, LfKCS3, from Physaria fendleri, a native HFA accumulator relative to camelina. The oil content and seed germination of the transgenic seeds also appeared normal compared to non-transgenics. LfKCS3 has been previously characterized to specifically elongate the hydroxylated ricinoleic acid to lesquerolic acid, the 20-carbon HFA found in lesquerella oil. The elongation reaction may facilitate the HFA flux from phosphatidylcholine (PC), the site of HFA formation, into the acyl-CoA pool for more efficient utilization in triacylglycerol (TAG) biosynthesis. This was demonstrated by increased HFA accumulation in TAG concurrent with reduced HFA content in PC during camelina seed development, and increased C20-HFA in HFA-TAG molecules. These effects of LfKCS3 thus may effectively relieve the bottleneck for HFA utilization in TAG biosynthesis and the feedback inhibition to fatty acid synthesis, result in higher HFA accumulation and restore oil content and seed viability.