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For decades, genetic engineering approaches to produce unusual fatty acids (UFAs) in crops has reached a bottleneck, including reduced seed oil production and seed vigor. Currently, plant models in the field of research are primarily used to investigate defects in oil production and seedling development, while the role of UFAs in embryonic developmental defects remains unknown. In this study, we developed a transgenic Arabidopsis plant model, in which the embryo exhibits severely wrinkled appearance owing to α-linolenic acid (ALA) accumulation. RNA-seq analysis in the defective embryo suggested that brassinosteroid synthesis, fatty acid (FA) synthesis, and photosynthesis were inhibited, while FA degradation, endoplasmic reticulum (ER) stress, and oxidative stress were activated. Lipidomics analysis showed that ultra-accumulated ALA is released from Phosphatidylcholine (PC) as a free fatty acid (FFA) in cell, inducing severe ER and oxidative stress. Furthermore, we identified that overexpression of lysophosphatidic acid acyltransferase 2 (LPAT2) rescued the defective phenotype. In the rescue line, the pool capacity of the Kennedy pathway was increased, and the esterification of ALA indirectly to triacylglycerol (TAG) was enhanced to avoid stress. This study provides a plant model that aids in understanding the molecular mechanism of embryonic developmental defects, and generate strategies to produce higher levels of UFAs.

Browning phenomena are ubiquitous in plant cell cultures that severely hamper scientific research and widespread application of plant cell cultures. Up to now, this problem still has not been well controlled due to the unclear browning mechanisms in plant cell cultures. In this paper, the mechanisms were investigated using two typical materials with severe browning phenomena, Taxus chinensis and Glycyrrhiza inflata cells. Our results illustrated that the browning is attributed to a physiological enzymatic reaction, and phenolic biosynthesis regulated by sugar plays a decisive role in the browning. Furthermore, to confirm the specific compounds which participate in the enzymatic browning reaction, transcriptional profile and metabolites of T. chinensis cells, and UV scanning and high-performance liquid chromatography-mass spectrometry (HPLC-MS) profile of the browning compounds extracted from the brown-turned medium were analyzed, flavonoids derived from phenylpropanoid pathway were found to be the main compounds, and myricetin and quercetin were deduced to be the main substrates of the browning reaction. Inhibition of flavonoid biosynthesis can prevent the browning occurrence, and the browning is effectively controlled via blocking flavonoid biosynthesis by gibberellic acid (GA3 ) as an inhibitor, which further confirms that flavonoids mainly contribute to the browning. On the basis above, a model elucidating enzymatic browning mechanisms in plant cell cultures was put forward, and effective control approaches were presented.

Dissolved oxygen (DO) is an important influencing factor in the process of aerobic microbial fermentation. Spinosad is an aerobic microbial-derived secondary metabolite. In our study, spinosad was used as an example to establish a DO strategy by multi-scale analysis, which included a reactor, cell and gene scales. We changed DO conditions that are related to the characteristics of cell metabolism (glucose consumption rate, biomass accumulation and spinosad production). Consequently, cell growth was promoted by maintaining DO at 40% in the first 24 h and subsequently increasing DO to 50% in 24 h to 96 h. In an in-depth analysis of the key enzyme genes (gtt, spn A, spn K and spn O), expression of spinosad and specific Adenosine Triphosphate (ATP), the spinosad yield was increased by regulating DO to 30% within 96 h to 192 h and then changing it to 25% in 192 h to 240 h. Under the four-phase DO strategy, spinosad yield increased by 652.1%, 326.1%, 546.8%, and 781.4% compared with the yield obtained under constant DO control at 50%, 40%, 30%, and 20% respectively. The proposed method provides a novel way to develop a precise DO strategy for fermentation.

Metabolomic and transcriptomic profiling data were obtained and integrated to elucidate the crucial network controls on taxol and its precursor biosynthesis during the taxane core functionalization within methyl jasmonate (MJ)-induced Taxus chinensis cells. Twelve metabolites were identified using liquid chromatography-electrospray ionization-mass spectrometry. These metabolites contain taxol (paclitaxel), baccatin III (B-III) and its analogs, a group structurally bearing multiple free hydroxyls (TAX), and another group of multiple acyl taxanes (MAT), including taxuyunnanine C (TC) and its analogs. The metabolomic profile showed a higher increase in TAX than in MAT. Particularly, the ratio of B-III and taxol to the total taxane content increased more significantly in TAX than in MAT. The MAT proportion did not significantly change, although they are predominant components in cell cultures compared with TAX. Quantitative real-time polymerase chain reaction (qRT-RCR) was used to determine the transcription level of 20 genes, among which 11 were reported responsible for taxol biosynthesis and 9 were obtained from our previous transcriptomic data. The total expression levels of hydroxylase after 24 h and 6 days were higher than those of acylase. The principal component analysis (PCA) results validated the metabolomic analysis data, indicating that hydroxylation was more crucial than acylation for controlling the flux toward TAX biosynthesis. Furthermore, the PCA contribution comparison showed that two undefined genes of OHX1 and ACX3 might have good potential in TAX upregulation and MAT downregulation. To the best of our knowledge, this study provides the first experimental evidence on the contribution of total hydroxylation to taxane biosynthesis. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:269–280, 2014

Although the regulation of taxol biosynthesis at the transcriptional level remains unclear, 10-deacetylbaccatin III-10 β-O-acetyl transferase (DBAT) is a critical enzyme in the biosynthesis of taxol. The 1740 bp fragment 5'-flanking sequence of the dbat gene was cloned from Taxus chinensis cells. Important regulatory elements needed for activity of the dbat promoter were located by deletion analyses in T. chinensis cells. A novel WRKY transcription factor, TcWRKY1, was isolated with the yeast one-hybrid system from a T. chinensis cell cDNA library using the important regulatory elements as bait. The gene expression of TcWRKY1 in T. chinensis suspension cells was specifically induced by methyl jasmonate (MeJA). Biochemical analysis indicated that TcWRKY1 protein specifically interacts with the two W-box (TGAC) cis-elements among the important regulatory elements. Overexpression of TcWRKY1 enhanced dbat expression in T. chinensis suspension cells, and RNA interference (RNAi) reduced the level of transcripts of dbat. These results suggest that TcWRKY1 participates in regulation of taxol biosynthesis in T. chinensis cells, and that dbat is a target gene of this transcription factor. This research also provides a potential candidate gene for engineering increased taxol accumulation in Taxus cell cultures.

As an alternative source in addition to fish oil, microbial production of docosahexaenoic acid has been recieved more and more attentions owing to their culture advantage. A unicellular eukaryotic microbe with high DHA production and capable of low temperature-adaptive growth was isolated from seawater and identified as Thraustochytriidae sp. Z105. The siginificant effect of temperature on cell growth and DHA synthesis by the strain was revealed. It could grow and produce DHA even at 4 °C, but hardly grow above 35 °C. Low temperature (15–25 °C) was favorable for formation of biomass, lipids and DHA, but DHA synthesis was completely blocked above 30 °C. Conditions for high level DHA production by Thraustochytriidae sp. Z105 in flask culture were optimized as follows: medium containing glucose 80 g/l, yeast extract 5.0 g/l, K2HPO4 · 3 H2O 1.0 g/l, MgSO4 · 7 H2O 0.5 g/l, seawater crystal 20 g/l, pH 6.0, liquid volume 30 ml/250 ml, temperature 20 °C, agitation speed of 200 r/min, and culture for 120 h. Under the optimal conditions, biomass of 16.72 g/l, total lipids of 5.35 g/l, DHA yield of 1.71 g/l (accounting for 32% of the total lipids) were achieved, respectively. In flask cluture level, the DHA productivity of Thraustochytriidae sp. Z105 was higher than most reported results, which suggested the wild type strain was a potential superior candidate for industrialization of DHA production. Moreover, the strain is an unique and valuable resource for investigation of the low temperature adaptive mechanism related to DHA synthesis. (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

BACKGROUND: The volatile oil of Lonicera japonica Thunb. is an edible natural perfume that is often used in foods, cigarettes and cosmetics. Chlorogenic acid, a major bioactive component of L. japonica, has received much attention recently owing to its antiviral, anticancer and anti-inflammatory activities. However, to date, few reports have focused on the variation in volatile oil and chlorogenic acid at different flowering stages of L. japonica. RESULTS: Obvious variations in components of the volatile oil were observed at six different developmental stages. The primary components of the volatile oil were linalool, linalool oxide, geraniol and α-terpineol, which reached their highest levels at the silver flowering stage. The highest levels of chlorogenic acid were found at the second white and complete white flowering stages. CONCLUSION: The results of this study indicate that the best times to harvest L. japonica flowers for volatile oil and chlorogenic acid extraction are the silver flowering stage and the second white or complete white flowering stage respectively. Copyright © 2009 Society of Chemical Industry

Lepidium meyenii Walp. (maca), a perennial herbaceous plant with narrow distribution in the Andean region, was cultivated by local residents as early as 1600 BC. It has high nutritional value and multiple medicinal effects. The plant source of maca is now scarce because maca products are becoming increasingly popular in the world as dietary supplements. This means that studies on the identification of maca materials are now urgent. In present work, natural plants with similar appearance or medicinal effects, including maca, radish (Raphanus sativus L.), oriental ginseng (Panax ginseng C. A. Mey.), American ginseng (Panax quinquefolium L.) were investigated. Their alkaloid extracts of the hypogeal parts were analysed by FTIR and TLC. The essential oils (steam distillates) were analysed by GC/MS. Through comparison of the characteristics of their spectra and chromatograms, it was found that the functional ingredient extracts of maca have unique FTIR, TLC and GC/MS behaviours. The secondary amide group (CH2NHCOCH2) and the phenyl structure in FTIR, the multiple spots at different RF values colourised by modified Dragendorff's reagent, and the characteristic peaks produced by the major essential oil components (e.g. phenylacetonitrile, benzaldehyde, 3-methoxyphenylacetonitrile) in GC/MS are distinct. These behaviours can be applied to the identification of maca or maca products in the market. Copyright © 2007 Society of Chemical Industry

Spores and oligosaccharide of a strain of Aspergillus niger, which had been isolated from the inner bark of Taxus chinensis, were used to treat T. chinensis leaves. The spores decreased the dry weight and chlorophyll content of the leaves, but increased their malondialdehyde content; oligosaccharide induced similar but more moderate changes. The changes in soluble protein, phenolic content, and peroxidase and phenylalanine ammonia-lyase activity were also analysed. Oligosaccharide significantly increased all these, whereas the spores had contrary effects. Results indicate that oligosaccharide, but not the fungal spores, elicited a defence reaction in T. chinensis. Possible reasons for these different effects on T. chinensis leaves in vitro are discussed.