Myrosinase isogenes in wasabi (Wasabia japonica Matsum) and their putative roles in glucosinolate metabolism.

BACKGROUND: Wasabi, a Brassicaceae member, is well-known for its unique pungent and hot flavor which is produced from glucosinolate (GSL) degradation. Myrosinase (MYR) is a principle enzyme catalyzing the primary conversion of GSLs to GSL hydrolysis products (GHPs) which is responsible for plant defense system and food quality. Due to the limited information in relation to MYRs present in wasabi (Wasabia japonica M.), this study aimed to identify the MYR isogenes in W. japonica and analyze their roles in relation to GSL metabolism. RESULTS: In results, WjMYRI-1 was abundantly expressed in all organs, whereas WjMYRI-2 showed only trace expression levels. WjMYRII was highly expressed in the aboveground tissues. Interestingly, WjMYRII expression was significantly upregulated by certain abiotic factors, such as methyl jasmonate (more than 40-fold in petioles and 15-fold in leaves) and salt (tenfold in leaves). Young leaves and roots contained 97.89 and 91.17 µmol‧g-1 of GSL, whereas less GSL was produced in mature leaves and petioles (38.36 and 44.79 µmol‧g-1, respectively). Similar pattern was observed in the accumulation of GHPs in various plant organs. Notably, despite the non-significant changes in GSL production, abiotic factors treated samples enhanced significantly GHP content. Pearson's correlation analysis revealed that WjMYRI-1 expression significantly correlated with GSL accumulation and GHP formation, suggesting the primary role of WjMYRI-1-encoding putative protein in GSL degradation. In contrast, WjMYRII expression level showed no correlation with GSL or GHP content, suggesting another physiological role of WjMYRII in stress-induced response. CONCLUSIONS: In conclusions, three potential isogenes (WjMYRI-1, WjMYRI-2, and WjMYRII) encoding for different MYR isoforms in W. japonica were identified. Our results provided new insights related to MYR and GSL metabolism which are important for the implications of wasabi in agriculture, food and pharmaceutical industry. Particularly, WjMYRI-1 may be primarily responsible for GSL degradation, whereas WjMYRII (clade II) may be involved in other regulatory pathways induced by abiotic factors.

Understanding the Impact of Nitrogen Availability: A Limiting Factor for Enhancing Fucoxanthin Productivity in Microalgae Cultivation.

This study aimed to investigate the regulation of fucoxanthin (FX) biosynthesis under various nitrogen conditions to optimize FX productivity in Phaeodactylum tricornutum. Apart from light, nitrogen availability significantly affects the FX production of microalgae; however, the underlying mechanism remains unclear. In batch culture, P. tricornutum was cultivated with normal (NN, 0.882 mM sodium nitrate), limited (LN, 0.22 mM), and high (HN, 8.82 mM) initial nitrogen concentrations in f/2 medium. Microalgal growth and photosynthetic pigment production were examined, and day 5 samples were subjected to fucoxanthin-chlorophyll a/c-binding protein (FCP) proteomic and transcriptomic analyses. The result demonstrated that HN promoted FX productivity by extending the exponential growth phase for higher biomass and FX accumulation stage (P1), showing a continuous increase in FX accumulation on day 6. Augmented FX biosynthesis via the upregulation of carotenogenesis could be primarily attributed to enhanced FCP formation in the thylakoid membrane. Key proteins, such as LHC3/4, LHCF8, LHCF5, and LHCF10, and key genes, such as PtPSY, PtPDS, and PtVDE, were upregulated under nitrogen repletion. Finally, the combination of low light and HN prolonged the P1 stage to day 10, resulting in maximal FX productivity to 9.82 ± 0.56 mg/L/day, demonstrating an effective strategy for enhancing FX production in microalgae cultivation.

Identification of drought-responsive phenolic compounds and their biosynthetic regulation under drought stress in Ligularia fischeri.

Ligularia fischeri, a leafy edible plant found in damp shady regions, has been used as an herbal medicine and is also consumed as a horticultural crop. In this study, we investigated the physiological and transcriptomic changes, especially those involved in phenylpropanoid biosynthesis, induced by severe drought stress in L. fischeri plants. A distinguishing characteristic of L. fischeri is a color change from green to purple due to anthocyanin biosynthesis. We chromatographically isolated and identified two anthocyanins and two flavones upregulated by drought stress using liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses in this plant for the first time. In contrast, all types of caffeoylquinic acids (CQAs) and flavonol contents were decreased under drought stress. Further, we performed RNA sequencing to examine the molecular changes in these phenolic compounds at the transcriptome level. In an overview of drought-inducible responses, we identified 2,105 hits for 516 distinct transcripts as drought-responsive genes. Moreover, differentially expressed genes (DEGs) associated with phenylpropanoid biosynthesis accounted for the greatest number of both up- and downregulated DEGs by Kyoto Encyclopedia of Genes and Genomes enrichment analysis. We identified 24 meaningful DEGs based on the regulation of phenylpropanoid biosynthetic genes. Potential drought-responsive genes included upregulated flavone synthase (LfFNS, TRINITY DN31661 c0 g1 i1) and anthocyanin 5-O-glucosyltransferase (LfA5GT1, TRINITY DN782 c0 g1 i1), which could contribute to the high levels of flavones and anthocyanins under drought stress in L. fischeri. In addition, the downregulated shikimate O-hydroxycinnamolytransferase (LfHCT, TRINITY DN31661 c0 g1 i1) and hydroxycinnamoyl-CoA quinate/shikimate transferase (LfHQT4, TRINITY DN15180 c0 g1 i1) genes led to a reduction in CQAs. Only one or two BLASTP hits for LfHCT were obtained for six different Asteraceae species. It is possible that the HCT gene plays a crucial role in CQAs biosynthesis in these species. These findings expand our knowledge of the response mechanisms to drought stress, particularly regarding the regulation of key phenylpropanoid biosynthetic genes in L. fischeri.

Nanoencapsulation enhances the bioavailability of fucoxanthin in microalga Phaeodactylum tricornutum extract.

The marine carotenoid fucoxanthin (FX) has various health benefits but suffers from poor bioavailability. We hypothesize that the bioavailability of FX in microalga Phaeodactylum tricornutum extract (PE) could be improved through nanoencapsulation. Here, we developed two types of nanoparticles: one consisting of alginate and casein (A-C-PE, 246 nm diameter, 79.6% encapsulation efficiency) and the other A-C-PE coated with chitosan (CS-A-C-PE, 258 nm, 78.1%). Both types of nanoparticles incorporating PE showed controlled FX release during simulated gastrointestinal digestion, as well as 1.8-fold improvement of membrane permeability in Caco-2/TC7 cells compared to non-encapsulated PE. Pharmacokinetic behavior of two FX metabolites (fucoxanthinol and amarouciaxanthin A) in mouse plasma was monitored after oral administration. The results showed that 31.8-332.1% more FX metabolites from the nanoparticles were absorbed into plasma than those from PE. In conclusion, encapsulation of PE in both types of nanoparticles significantly promoted the bioavailability of FX.

Detection of DNA damage in oocytes of small ovarian follicles following phosphoramide mustard exposures of cultured rodent ovaries in vitro.

Healthy oocytes are critical for producing healthy children, but little is known about whether or not oocytes have the capacity to identify and recover from injury. Using a model ovotoxic alkylating drug, cyclophosphamide (CPA), and its active metabolite, phosphoramide mustard (PM), we previously showed that PM (≥3µM) caused significant follicle loss in postnatal day 4 (PND4) mouse ovaries in vitro. We now investigate whether PM induces DNA damage in oocytes, examining histone H2AX phosphorylation (γH2AX), a marker of DNA double-strand breaks (DSBs). Exposure of cultured PND4 mouse ovaries to 3 and 0.1µM PM induced significant losses of primordial and small primary follicles, respectively. PM-induced γH2AX was observed predominantly in oocytes, in which foci of γH2AX staining increased in a concentration-dependent manner and peaked 18-24h after exposure to 3-10µMPM. Numbers of oocytes with ≥5 γH2AX foci were significantly increased both 1 and 8days after exposure to ≥1µMPM compared to controls. Inhibiting the kinases that phosphorylate H2AX significantly increased follicle loss relative to PM alone. In adult mice, CPA also induced follicle loss in vivo. PM also significantly decreased primordial follicle numbers (≥30µM) and increased γH2AX foci (≥3µM) in cultured PND4 Sprague-Dawley rat ovaries. Results suggest oocytes can detect PM-induced damage at or below concentrations which cause significant follicle loss, and there are quantitative species-specific differences in sensitivity. Surviving oocytes with DNA damage may represent an increased risk for fertility problems or unhealthy offspring.

SR-BI, CD36, and caveolin-1 contribute positively to cholesterol efflux in hepatic cells.

In non-hepatic cells, scavenger receptor class B type I (SR-BI), cluster of differentiation 36 (CD36), and caveolin-1 were described as mediators of cholesterol efflux, the first step of reverse cholesterol transport (RCT). Stable transformants of HepG2 cells overexpressing SR-BI, CD36, or caveolin-1 were generated, as well as cells overexpressing both caveolin-1 and SR-BI or caveolin-1 and CD36 in order to address the effect of caveolin-1 on both receptor activities. These cells were analyzed for their ability to efflux cholesterol to HDL(3). Our results show that overexpressing SR-BI, CD36, or caveolin-1 increases cholesterol efflux by 106, 92, and 48%, respectively. Moreover, the dual overexpressions of caveolin-1 and SR-BI or caveolin-1 and CD36 lead to a more prominent increase in cholesterol efflux. Studies were also conducted with primary cultures of SR-BI knockout (KO), CD36 KO, and SR-BI/CD36 double-KO (dKO) mice. SR-BI KO and SR-BI/CD36 dKO hepatic cells show 41 and 56% less cholesterol efflux, respectively, than normal hepatic cells. No significant difference was observed between the efflux of normal and CD36 KO cells. The difference between the role of human and murine CD36 correlated with the absence of CD36 dimers in mouse caveolae/rafts. Overall, our results show that SR-BI is clearly involved in cholesterol efflux in mouse and human hepatic cells, while CD36 plays a significant role in human cells.

Expression of caveolin-1 in hepatic cells increases oxidized LDL uptake and preserves the expression of lipoprotein receptors.

Oxidized LDL (OxLDL) that are positively associated with the risk of developing cardiovascular diseases are ligands of scavenger receptor-class B type I (SR-BI) and cluster of differentiation-36 (CD36) which can be found in caveolae. The contribution of these receptors in human hepatic cell is however unknown. The HepG2 cell, a human hepatic parenchymal cell model, expresses these receptors and is characterized by a very low level of caveolin-1. Our aim was to define the contribution of human CD36, SR-BI, and caveolin-1 in the metabolism of OxLDL in HepG2 cells and conversely the effects of OxLDL on the levels/localization of these receptors. By comparing mildly (M)- and heavily (H)-OxLDL metabolism between control HepG2 cells and HepG2 cells overexpressing CD36, SR-BI, or caveolin-1, we found that (1) CD36 increases M- and H-OxLDL-protein uptake; (2) SR-BI drives M-OxLDL through a degradation pathway at the expense of the cholesterol ester (CE) selective uptake pathway; (3) caveolin-1 increases M- and H-OxLDL-protein uptake and decreases CE selective uptake from M-OxLDL. Also, incubation with M- or H-OxLDL decreases the levels of SR-BI and LDL-receptor in control HepG2 cells which can be overcome by caveolin-1 expression. In addition, OxLDL move CD36 from low to high buoyant density membrane fractions, as well as caveolin-1 in cells overexpressing this protein. Thus, hepatic caveolin-1 expression has significant effects on OxLDL metabolism and on lipoprotein receptor levels.

Differential abilities of mouse liver parenchymal and nonparenchymal cells in HDL and LDL (native and oxidized) association and cholesterol efflux.

The aim of this study was to quantify the abilities of mouse liver parenchymal and nonparenchymal cells with respect to (i) cholesteryl ester (CE) selective uptake from low-density lipoproteins (LDL), oxidized LDL (OxLDL), and high-density lipoprotein (HDL); and (ii) their free cholesterol efflux to HDL. The preparations of cells were incubated with lipoproteins labelled either in protein with iodine-125 or in CE with 3H-cholesterol oleate, and lipoprotein-protein and lipoprotein-CE associations were measured. The associations of LDL-protein and LDL-CE with nonparenchymal cells were 5- and 2-fold greater, respectively, than with parenchymal cells. However, in terms of CE-selective uptake (CE association minus protein association) both types of cell were equivalent. Similar results were obtained with OxLDL, but both types of cell showed higher abilities in OxLDL-CE than in LDL-CE selective uptake (on average by 3.4-fold). The association of HDL-protein with nonparenchymal cells was 3x that with parenchymal cells; however, nonparenchymal cells associated 45% less HDL-CE. Contrary to parenchymal cells, nonparenchymal cells did not show HDL-CE selective uptake activity. Thus parenchymal cells selectively take CE from the 3 types of lipoproteins, whereas nonparenchymal cells exert this function only on LDL and OxLDL. Efflux was 3.5-fold more important in nonparenchymal than in parenchymal cells.

Opposite effect of caveolin-1 in the metabolism of high-density and low-density lipoproteins.

Receptors of the scavenger class B family were reported to be localized in caveolae, the cell surface microdomains rich in free cholesterol and glycosphyngolipids, which are characterized by the presence of caveolin-1. Parenchymal hepatic and hepatoma HepG2 cells express very low levels of caveolin-1. In the present study, stable transformants of HepG2 cells expressing caveolin-1 were generated to address the effect of caveolin-1 on receptor activity. Compared to normal cells, these cells show higher (125)I-bovine serum albumin (BSA) uptake and cholesterol efflux, two indicators of functional caveolae. By immunoprecipitation, cell fractionation and confocal analyses, we found that caveolin-1 is well colocalized with the cluster of differentiation-36 (CD36) and the low-density lipoprotein (LDL) receptor (LDLr) but to a lesser extent with the scavenger receptor class B type I (SR-BI) in HepG2 cells expressing caveolin-1. However, caveolin-1 expression favors the dimerization of SR-BI. Two clones of cells expressing caveolin-1 were investigated for their lipoprotein metabolism activity. Compared to normal cells, these cells show a 71-144% increase in (125)I-LDL degradation. The analysis of the cholesteryl esters (CE)-selective uptake (CE association minus protein association) revealed that the expression of caveolin-1 in HepG2 cells decreases by 59%-73% LDL-CE selective uptake and increases high-density lipoprotein (HDL)-CE selective uptake by 44%-66%. We conclude that the expression of caveolin-1 in HepG2 cells moves the balance of LDL degradation/CE selective uptake towards degradation and favors HDL-CE selective uptake. Thus, in the normal hepatic parenchymal situation where caveolin-1 is poorly expressed, LDL-CE selective uptake is the preferred pathway.