Eriodictyol can modulate cellular auxin gradients to efficiently promote in vitro cotton fibre development.
ABSTRACT: BACKGROUND:Flavonoids have essential roles in flower pigmentation, fibre development and disease resistance in cotton. Previous studies show that accumulation of naringenin in developing cotton fibres significantly affects fibre growth. This study focused on determining the effects of the flavonoids naringenin, dihydrokaempferol, dihydroquerectin and eriodictyol on fibre development in an in vitro system. RESULTS:20??M eriodictyol treatment produced a maximum fibre growth, in terms of fibre length and total fibre units. To gain insight into the associated transcriptional regulatory networks, RNA-seq analysis was performed on eriodictyol-treated elongated fibres, and computational analysis of differentially expressed genes revealed that carbohydrate metabolism and phytohormone signaling pathways were differentially modulated. Eriodictyol treatment also promoted the biosynthesis of quercetin and dihydroquerectin in ovules and elongating fibres through enhanced expression of genes encoding chalcone isomerase, chalcone synthase and flavanone 3-hydroxylase. In addition, auxin biosynthesis and signaling pathway genes were differentially expressed in eriodictyol-driven in vitro fibre elongation. In absence of auxin, eriodictyol predominantly enhanced fibre growth when the localized auxin gradient was disrupted by the auxin transport inhibitor, triiodobenzoic acid. CONCLUSION:Eriodictyol was found to significantly enhance fibre development through accumulating and maintaining the temporal auxin gradient in developing unicellular cotton fibres.
Project description:The health benefits of flavonoids for humans are increasingly attracting attention. Because the extraction of high-purity flavonoids from plants presents a major obstacle, interest has emerged in biosynthesizing them using microbial hosts. Eriodictyol is a flavonoid with anti-inflammatory and antioxidant activities. Its efficient synthesis has been hampered by two factors: the poor expression of cytochrome P450 and the low intracellular malonyl coenzyme A (malonyl-CoA) concentration in Escherichia coli. To address these issues, a truncated plant P450 flavonoid, flavonoid 3'-hydroxylase (tF3'H), was functionally expressed as a fusion protein with a truncated P450 reductase (tCPR) in E. coli. This allowed the engineered E. coli to produce eriodictyol from l-tyrosine by simultaneously coexpressing the fusion protein with tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). In addition, metabolic engineering was employed to enhance the availability of malonyl-CoA so as to achieve a new metabolic balance and rebalance the relative expression of genes to enhance eriodictyol accumulation. This approach made the production of eriodictyol 203% higher than that in the control strain. By using these strategies, the production of eriodictyol from l-tyrosine reached 107 mg/liter. The present work offers an approach to the efficient synthesis of other hydroxylated flavonoids from l-tyrosine or even glucose in E. coli.
Project description:Natural plant-based flavonoids have drawn significant attention as dietary supplements due to their potential health benefits, including anti-cancer, anti-oxidant and anti-asthmatic activities. Naringenin, pinocembrin, eriodictyol and homoeriodictyol are classified as (2S)-flavanones, an important sub-group of naturally occurring flavonoids, with wide-reaching applications in human health and nutrition. These four compounds occupy a central position as branch point intermediates towards a broad spectrum of naturally occurring flavonoids. Here, we report the development of Escherichia coli production chassis for each of these key gatekeeper flavonoids. Selection of key enzymes, genetic construct design and the optimization of process conditions resulted in the highest reported titers for naringenin (484 mg/l), improved production of pinocembrin (198 mg/l) and eriodictyol (55 mg/l from caffeic acid), and provided the first example of in vivo production of homoeriodictyol directly from glycerol (17 mg/l). This work provides a springboard for future production of diverse downstream natural and non-natural flavonoid targets.
Project description:Cytochrome P450 monooxygenase constitutes a significant group of oxidative enzymes that can introduce an oxygen atom in a high regio- and stereo-selectivity mode. We used the Bacillus megaterium cytochrome P450 BM3 (CYP450 BM3) and its variants namely mutant 13 (M13) and mutant 15 (M15) for the hydroxylation of diverse class of flavonoids.Among 20 flavonoids, maximum seven flavonoids were hydroxylated by the variants while none of these molecules were accepted by CYP450 BM3 in in vitro reaction. Moreover, M13 exhibited higher conversion of substrates than M15 and CYP450 BM3 enzymes. We found that M13 carried out regiospecific 3'-hydroxylation reaction of naringenin with the highest conversion among all the tested flavonoids. The apparent K m and k cat values of M13 for naringenin were 446 µM and 1.955 s(-1), respectively. In whole-cell biotransformation experiment with 100 µM of naringenin in M9 minimal medium with 2 % glucose in shake flask culture, M13 showed 2.14- and 13.96-folds higher conversion yield in comparison with M15 (16.11 %) and wild type (2.47 %). The yield of eriodictyol was 46.95 µM [~40.7 mg (13.5 mg/L)] in a 3-L volume lab scale fermentor at 48 h in the same medium exhibiting approximately 49.81 % conversion of the substrate. In addition, eriodictyol exhibited higher antibacterial and anticancer potential than naringenin, flavanone and hesperetin.We elucidated that eriodictyol being produced from naringenin using recombinant CYP450 BM3 and its variants from B. megaterium, which shows an approach for the production of important hydroxylated compounds of various polyphenols that may span pharmaceutical industries.
Project description:Nutraceutical compounds as plant flavonoids play an important role in prevention and modulation of diverse heath conditions, as they exert interesting antifungal, antibacterial, antioxidant, and antitumor effects. They also possess anti-inflammatory activities in arthritis, cardiovascular disease or neurological diseases, as well as modulatory effects on the CYP450 activity on diverse drugs. Most flavonoids are bioactive molecules of plant origin, but their industrial production is sometimes hindered due to reasons as low concentration in the plant tissues, presence in only some species or as a complex mixture or inactive glycosides in plant vacuolae. In this work, we describe the de novo biosynthesis of two important flavones, apigenin and luteolin, and one known flavanone, eriodictyol. Their plant biosynthetic pathways have been reconstructed for heterologous expression in Streptomyces albus, an actinomycete bacterium manageable at industrial production level. Also, production levels for apigenin have been improved by feeding with naringenin precursor, and timing for settlement of secondary metabolism has been advanced by spore conditioning. In the cases of eriodictyol and luteolin, their production in this important type of biotechnology-prone bacteria, the actinomycetes, had not been described in the literature yet.
Project description:Three different flavonoids-naringenin, quercetin and kaempferol-accumulate in root galls of Arabidopsis thaliana after infection with the obligate biotrophic pathogen Plasmodiophora brassicae. In addition, high-performance liquid chromatography and thin layer chromatography analysis indicated that these flavonoids and their glycosides were induced in galls rather than in healthy roots. The transcripts of selected genes involved in the biosynthesis of flavonoids were up-regulated during the time course of the disease. Some, such as chalcone synthase and chalcone isomerase, were up-regulated at both times investigated in this study, whereas up-regulation was observed only at later times for others, such as a flavonol synthase-like gene. Plants with mutations in different flavonoid biosynthesis genes were slightly more tolerant to clubroot at low infection pressure. However, flavonoid treatment of either leaves or roots did not reduce gall development. The possibility that flavonoids might influence auxin levels by regulating auxin transport or auxin degradation in roots was investigated by measuring auxin levels and response in roots of flavonoid-deficient mutants and the wild-type after inoculation with P. brassicae, as well as the antioxidative potential of flavonoids in the peroxidase-catalysed degradation of indole-3-acetic acid. In addition, the auxin transport rate from the shoots to the roots was measured in infected wild-type or flavonoid mutant plants compared with controls. In conclusion, our results indicate a role of flavonoids in the modulation of auxin efflux in root galls.
Project description:The biosynthesis of flavonoids and anthocyanidins has been exclusively investigated in angiosperms but largely unknown in ferns. This study integrated metabolomics and transcriptome to analyze the fronds from different development stages (S1 without spores and S2 with brown spores) of Cyclosorus parasiticus. About 221 flavonoid and anthocyanin metabolites were identified between S1 and S2. Transcriptome analysis revealed several genes encoding the key enzymes involved in the biosynthesis of flavonoids, and anthocyanins were upregulated in S2, which were validated by qRT-PCR. Functional characterization of two chalcone synthases (CpCHS1 and CpCHS2) indicated that CpCHS1 can catalyze the formation of pinocembrin, naringenin, and eriodictyol, respectively; however, CpCHS2 was inactive. The crystallization investigation of CpCHS1 indicated that it has a highly similar conformation and shares a similar general catalytic mechanism to other plants CHSs. And by site-directed mutagenesis, we found seven residues, especially Leu199 and Thr203 that are critical to the catalytic activity for CpCHS1.
Project description:Brown cotton fibres are the most widely used naturally coloured raw materials for the eco-friendly textile industry. Previous studies have indicated that brown fibre pigments belong to proanthocyanidins (PAs) or their derivatives, and fibre coloration is negatively associated with cotton productivity and fibre quality. To date, the molecular basis controlling the biosynthesis and accumulation of brown pigments in cotton fibres is largely unknown. In this study, based on expressional and transgenic analyses of cotton homologs of ArabidopsisPA regulator TRANSPARENT TESTA 2 (TT2) and fine-mapping of the cotton dark-brown fibre gene (Lc1), we show that a TT2 homolog, GhTT2-3A, controls PA biosynthesis and brown pigmentation in cotton fibres. We observed that GhTT2-3A activated GhbHLH130D, a homolog of ArabidopsisTT8, which in turn synergistically acted with GhTT2-3A to activate downstream PA structural genes and PA synthesis and accumulation in cotton fibres. Furthermore, the up-regulation of GhTT2-3A in fibres at the secondary wall-thickening stage resulted in brown mature fibres, and fibre quality and lint percentage were comparable to that of the white-fibre control. The findings of this study reveal the regulatory mechanism controlling brown pigmentation in cotton fibres and demonstrate a promising biotechnological strategy to break the negative linkage between coloration and fibre quality and/or productivity.
Project description:BACKGROUND: Cotton is the dominant source of natural textile fibre and a significant oil crop. Cotton fibres, produced by certain species in the genus Gossypium, are seed trichomes derived from individual cells of the epidermal layer of the seed coat. Cotton fibre development is delineated into four distinct and overlapping developmental stages: fibre initiation, elongation, secondary wall biosynthesis and maturation. SCOPE: Recent advances in gene expression studies are beginning to provide new insights into a better understanding of early events in cotton fibre development. Fibre cell development is a complex process involving many pathways, including various signal transduction and transcriptional regulation components. Several analyses using expressed sequence tags and microarray have identified transcripts that preferentially accumulate during fibre development. These studies, as well as complementation and overexpression experiments using cotton genes in arabidopsis and tobacco, indicate some similar molecular events between trichome development from the leaf epidermis and fibre development from the ovule epidermis. Specifically, MYB transcription factors regulate leaf trichome development in arabidopsis and may regulate seed trichome development in cotton. In addition, transcript profiling and ovule culture experiments both indicate that several phytohormones and other signalling pathways mediate cotton fibre development. Auxin and gibberellins promote early stages of fibre initiation; ethylene- and brassinosteroid-related genes are up-regulated during the fibre elongation phase; and genes associated with calmodulin and calmodulin-binding proteins are up-regulated in fibre initials. Additional genomic data, mutant and functional analyses, and genome mapping studies promise to reveal the critical factors mediating cotton fibre cell development.
Project description:High-quality cotton fibre equates to a more comfortable textile. Fibre length is an important index of fibre quality. Hydrogen peroxide (H2O2) acts as a signalling molecule in the regulation of fibre elongation. Results from in vitro ovule culture suggest that the alteration of fibre cell H2O2 levels affects fibre development. Ascorbate peroxidase (APX) is an important reactive oxygen species (ROS) scavenging enzyme, and we found that GhAPX1AT/DT encoded one member of the previously unrealized group of cytosolic APXs (cAPXs) that were preferentially expressed during the fibre elongation stage. Transgenic cottons with up- and down-regulation of GhAPX1AT/DT were generated to control fibre endogenous levels of H2O2 Suppression of all cAPX (IAO) resulted in a 3.5-fold increase in H2O2 level in fibres and oxidative stress, which significantly suppressed fibre elongation. The fibre length of transgenic lines with over-expression or specific down-regulation of GhAPX1AT/DT did not show any obvious change. However, the fibres in the over-expression lines exhibited higher tolerance to oxidative stress. Differentially expressed genes (DEGs) in fibres at 10 days post-anthesis (DPA) of IAO lines identified by RNA-seq were related to redox homeostasis, signalling pathways, stress responses and cell wall synthesis, and the DEGs that were up-regulated in IAO lines were also up-regulated in the 10 DPA and 20 DPA fibres of wild cotton compared with domesticated cotton. These results suggest that optimal H2O2 levels and redox state regulated by cytosolic APX are key mechanisms regulating fibre elongation, and dysregulation of the increase in H2O2 induces oxidative stress and results in shorter fibres by initiating secondary cell wall-related gene expression.
Project description:The present study was aimed to evaluate the protective effects of six different polyphenols against benzo(a)pyrene (BaP)-induced cytotoxicity in Caco-2 cells. The results shows that treatment of quinic acid, ferulic acid, homovanillic acid and BaP decreased the cell viability, whereas only co-treatment of 20 µM eriodictyol and naringenin reduced BaP-induced cytotoxicity, including cell apoptosis, cell cycle change and oxidative stress. Moreover, all results show that the inhibitory effect of eriodictyol was better than that of naringenin. Further, the potential protective mechanism of eriodictyol against BaP-induced toxicity was investigated by proteomics. We found that the genetic information processing pathway involves in 80 differentially expressed proteins (DEPs) showed the highest proportion and number of total proteins. Finally, eriodictyol inhibited BaP-induced cytotoxicity by regulating the expression of key proteins such as RPA2, SNRPA, RAD23B, NUP155 and AARS in transcription and translation. Overall, our results provide a new perspective for polyphenols inhibiting BaP-induced carcinogenesis.