Assessing Silicon-Mediated Growth Performances in Contrasting Rice Cultivars under Salt Stress.

Silicon (Si) application has great potential to improve salt tolerance in a variety of crop plants. However, it is unclear how Si influences the responses of contrasting rice cultivars when exposed to excessive salt. Here, we investigated the functions of Si in alleviating the negative effects of salt stress on two contrasting rice cultivars, namely BRRI dhan48 (salt-sensitive) and Binadhan-10 (salt-tolerant). Rice seedlings was pre-treated with three doses of Si (as silicic acid; 0, 1 and 2 mM) for 14 days at one-day interval before being exposed to salt stress (10 dSm-1) in a sustained water bath system. The results demonstrated that the seedlings of BRRI dhan48 and Binadhan-10, respectively exhibited substantial reductions in shoot height (16 and 9%), shoot fresh weight (64 and 43%) and shoot dry weight (50 and 39%) under salinity. Intriguingly, BRRI dhan48 pre-treated with 1 and 2 mM Si, respectively, showed a higher increase in shoot height (SH) (by 25.90 and 26.08%) as compared with Binadhan-10 (by 3 and 8%) under salt stress compared with their respective controls. Data revealed that a comparatively higher improvement in the growth performances of the salt-induced Si pre-treated BRRI dhan48 than that of Binadhan-10. For example, 1 and 2 mM of Si treatments significantly attributed to elevated leaf relative water content (RWC) (13 and 22%), proline (138 and 165%), chlorophyll a (42 and 44%), chlorophyll b (91 and 72%), total chlorophyll (58 and 53%) and carotenoids (33 and 29%), and recovery in the reductions of electrolyte leakage (13 and 21%), malondialdehyde content (23 and 30%) and shoot Na+/K+ ratio (22 and 52%) in BRRI dhan48 compared with Si-untreated control plants under salt stress. In addition, we found salt-tolerant Binadhan-10 also had enhanced RWC (9 and 19%), proline (12 and 26%) with pre-treatment with 1 and 2 mM of Si, respectively, under salt stress, while no significant differences were noticed in the case of photosynthetic pigments and Na+/K+ ratio. Our results showed that Si supplementation potentiated higher salt-tolerance ability in the salt-sensitive BRRI dhan48 as compared with salt-tolerant Binadhan-10. Thus, Si application could be highly beneficial in the growth recovery of the salinity-affected salt-sensitive high yielding rice cultivars in the saline-prone areas.

Enhanced triterpene accumulation in Panax ginseng hairy roots overexpressing mevalonate-5-pyrophosphate decarboxylase and farnesyl pyrophosphate synthase.

To elucidate the function of mevalonate-5-pyrophosphate decarboxylase (MVD) and farnesyl pyrophosphate synthase (FPS) in triterpene biosynthesis, the genes governing the expression of these enzymes were transformed into Panax ginseng hairy roots. All the transgenic lines showed higher expression levels of PgMVD and PgFPS than that by the wild-type control. Among the hairy root lines transformed with PgMVD, M18 showed the highest level of transcription compared to the control (14.5-fold higher). Transcriptions of F11 and F20 transformed with PgFPS showed 11.1-fold higher level compared with control. In triterpene analysis, M25 of PgMVD produced 4.4-fold higher stigmasterol content (138.95 µg/100 mg, dry weight [DW]) than that by the control; F17 of PgFPS showed the highest total ginsenoside (36.42 mg/g DW) content, which was 2.4-fold higher compared with control. Our results indicate that metabolic engineering in P. ginseng was successfully achieved through Agrobacterium rhizogenes-mediated transformation and that the accumulation of phytosterols and ginsenosides was enhanced by introducing the PgMVD and PgFPS genes into the hairy roots of the plant. Our results suggest that PgMVD and PgFPS play an important role in the triterpene biosynthesis of P. ginseng.

Enhancement of anti-inflammatory activity of Aloe vera adventitious root extracts through the alteration of primary and secondary metabolites via salicylic acid elicitation.

Aloe vera (Asphodeloideae) is a medicinal plant in which useful secondary metabolites are plentiful. Among the representative secondary metabolites of Aloe vera are the anthraquinones including aloe emodin and chrysophanol, which are tricyclic aromatic quinones synthesized via a plant-specific type III polyketide biosynthesis pathway. However, it is not yet clear which cellular responses can induce the pathway, leading to production of tricyclic aromatic quinones. In this study, we examined the effect of endogenous elicitors on the type III polyketide biosynthesis pathway and identified the metabolic changes induced in elicitor-treated Aloe vera adventitious roots. Salicylic acid, methyl jasmonate, and ethephon were used to treat Aloe vera adventitious roots cultured on MS liquid media with 0.3 mg/L IBA for 35 days. Aloe emodin and chrysophanol were remarkably increased by the SA treatment, more than 10-11 and 5-13 fold as compared with untreated control, respectively. Ultra-performance liquid chromatography-electrospray ionization mass spectrometry analysis identified a total of 37 SA-induced compounds, including aloe emodin and chrysophanol, and 3 of the compounds were tentatively identified as tricyclic aromatic quinones. Transcript accumulation analysis of polyketide synthase genes and gas chromatography mass spectrometry showed that these secondary metabolic changes resulted from increased expression of octaketide synthase genes and decreases in malonyl-CoA, which is the precursor for the tricyclic aromatic quinone biosynthesis pathway. In addition, anti-inflammatory activity was enhanced in extracts of SA-treated adventitious roots. Our results suggest that SA has an important role in activation of the plant specific-type III polyketide biosynthetic pathway, and therefore that the efficacy of Aloe vera as medicinal agent can be improved through SA treatment.

Cloning and characterization of a cDNA encoding calcium/calmodulin-dependent glutamate decarboxylase from Scutellaria baicalensis.

Gamma-aminobutyric acid (GABA), synthesized by glutamate decarboxylase (GAD), plays an important role in plants. To study the molecular mechanism of GAD regulation and to examine the levels of GABA in Scutellaria baicalensis, we isolated cDNA clones (SbGAD1 and 2) encoding GAD from S. baicalensis. The open reading frames of SbGADI and 2 were 1,503 and 1,494 bp long and had 450 and 497 amino acid residues, respectively. Quantitative real-time RT-PCR analysis was performed to show the variation of transcript levels among different organs of S. baicalensis. Transcript levels of SbGAD1 and 2 were highest in the root and flower, respectively. The GABA content of different parts (ranked in descending order) was as follows: leaf > flower > stem > root. We concluded that the expression pattern of SbGAD1 and 2 did not match the accumulation pattern of GABA in different organs. We presume that GABA biosynthesis might be more controlled by SbGAD2 than SbGADI. These data will aid in future studies that seek to understand the mechanisms underlying GABA biosynthesis, an important amino acid that is synthesized by the GAD enzyme. To explain adequately the GABA biosynthesis mechanisms in S. baicalensis, the enzyme activities of SbGAD1 and 2 should be determined in the near future.

Neuroprotective effects of Lycium chinense Miller against rotenone-induced neurotoxicity in PC12 cells.

Rotenone, an inhibitor of mitochondrial complex I, has been widely regarded as a neurotoxin because it induces a Parkinson's disease-like syndrome. The fruit and root bark of Lycium chinense Miller have been used as traditional medicines in Asia to treat neurodegenerative diseases. In this study, we examined the neuroprotective effects of Lycium chinense Miller extracts in rotenone-treated PC12 cells. Treatment with rotenone reduced PC12 cell viability and cellular ATP levels. Conversely, caspase 3/7 activity, the ratio of Bax:Bcl-2 expression levels, mitochondrial superoxide level, and intracellular calcium (Ca(2+)) concentration were elevated. Pretreatment with Lycium chinense Miller extracts significantly increased cell viability and ATP levels. Additionally, they attenuated caspase activation, mitochondrial membrane depolarization and mitochondrial superoxide production. Moreover, confocal microscopy showed that the mitochondrial staining pattern was restored from that of extracts treated cells and that the increase in intracellular Ca (2+) level was blunted by treatment with the extracts. Our results suggest that Lycium chinense Miller extracts may have the possible beneficial effects in Parkinson's disease by attenuating rotenone induced toxicity.

Metabolomic analysis and phenylpropanoid biosynthesis in hairy root culture of tartary buckwheat cultivars.

Buckwheat, Fagopyrum tataricum Gaertn., is an important medicinal plant, which contains several phenolic compounds, including one of the highest content of rutin, a phenolic compound with anti-inflammatory properties. An experiment was conducted to investigate the level of expression of various genes in the phenylpropanoid biosynthetic pathway to analyze in vitro production of anthocyanin and phenolic compounds from hairy root cultures derived from 2 cultivars of tartary buckwheat (Hokkai T8 and T10). A total of 47 metabolites were identified by gas chromatography-time-of-flight mass spectrometry (GC-TOFMS) and subjected to principal component analysis (PCA) in order to fully distinguish between Hokkai T8 and T10 hairy roots. The expression levels of phenylpropanoid biosynthetic pathway genes, through qRT-PCR, showed higher expression for almost all the genes in T10 than T8 hairy root except for FtF3'H-2 and FtFLS-2. Rutin, quercetin, gallic acid, caffeic acid, ferulic acid, 4-hydroxybenzoic acid, and 2 anthocyanin compounds were identified in Hokkai T8 and T10 hairy roots. The concentration of rutin and anthocyanin in Hokkai T10 hairy roots of tartary buckwheat was several-fold higher compared with that obtained from Hokkai T8 hairy root. This study provides useful information on the molecular and physiological dynamic processes that are correlated with phenylpropanoid biosynthetic gene expression and phenolic compound content in F. tataricum species.

Protection from antimycin A-induced mitochondrial dysfunction by Nelumbo nucifera seed extracts.

Antimycin A (AMA) damages the mitochondria through inhibition of mitochondrial electron transport. In this study, exposure of L6 rat skeletal muscle cells to AMA induced a decrease in ATP content, followed by a decrease in mitochondrial membrane potential, leading to apoptosis. We evaluated the protective effects of water and ethanol extracts of Nelumbo nucifera seeds on L6 cells with AMA-induced oxidative stress. We found that the extracts reduced cellular apoptosis; preserved the mitochondrial membrane potential; protected mitochondrial ATP production; inhibited p53, Bax, and caspase 3 activities; and induced Bcl-2 production. Our results suggested that AMA induced apoptosis in L6 cells via impairment of mitochondrial function. N. nucifera extracts protected the cells from this mitochondria-mediated cell death.

Glucosinolate biosynthesis in hairy root cultures of broccoli (Brassica oleracea var. italica).

Here we present previously unreported glucosinolate production by hairy root cultures of broccoli (B. oleracea var. italica). Growth media greatly influenced the growth and glucosinolate content of hairy root cultures of broccoli. Seven glucosinolates, glucoraphanin, gluconapin, glucoerucin, glucobrassicin, 4-methoxyglucobrassicin, gluconasturtiin, and neoglucobrassicin, were identified by analysis of the broccoli hairy root cultures. Both half and full strength B5 and SH media enabled the highest accumulation of glucosinolates. In most cases, the levels of glucosinolates were higher in SH and BS media. Among the 7 glucosinolates, the accumulation of neoglucobrassicin was very high, irrespective of growth medium. The neoglucobrassicin content was 7.4-fold higher in SH medium than 1/2 MS, in which its level was the lowest. The 1/2 B5 medium supported the production of the highest amounts of glucobrassicin and 4-methoxyglucobrassicin, the levels for which were 36.2- and 7.9- fold higher, respectively, than their lowest content in 1/2 MS medium. The 1/2 SH medium enabled the highest accumulation of glucoraphanin and gluconapin in the broccoli hairy root cultures, whose levels were 1.8- and 4.6-fold higher, respectively, than their lowest content in 1/2 MS medium. Our results suggest that hairy root cultures of broccoli could be a valuable alternative approach for the production of glucosinolate compounds.

Scutellaria baicalensis Extracts and Flavonoids Protect Rat L6 Cells from Antimycin A-Induced Mitochondrial Dysfunction.

Antimycin A (AMA) damages mitochondria by inhibiting mitochondrial electron transport and can produce reactive oxygen species (ROS). ROS formation, aging, and reduction of mitochondrial biogenesis contribute to mitochondrial dysfunction. The present study sought to investigate extracts of Scutellaria baicalensis and its flavonoids (baicalin, baicalein, and wogonin), whether they could protect mitochondria against oxidative damage. The viability of L6 cells treated with AMA increased in the presence of flavonoids and extracts of S. baicalensis. ATP production decreased in the AMA treated group, but increased by 50% in cells treated with flavonoids (except wogonin) and extracts of S. baicalensis compared to AMA-treated group. AMA treatment caused a significant reduction (depolarized) in mitochondrial membrane potential (MMP), whereas flavonoid treatment induced a significant increase in MMP. Mitochondrial superoxide levels increased in AMA treated cells, whereas its levels decreased when cells were treated with flavonoids or extracts of S. baicalensis. L6 cells treated with flavonoids and extracts of S. baicalensis increased their levels of protein expression compared with AMA-treated cells, especially water extracts performed the highest levels of protein expression. These results suggest that the S. baicalensis extracts and flavonoids protect against AMA-induced mitochondrial dysfunction by increasing ATP production, upregulating MMP, and enhancing mitochondrial function.

Metabolic profiling of glucosinolates, anthocyanins, carotenoids, and other secondary metabolites in kohlrabi (Brassica oleracea var. gongylodes).

We profiled and quantified glucosinolates, anthocyanins, carotenoids, and other secondary metabolites in the skin and flesh of pale green and purple kohlrabis. Analysis of these distinct kohlrabis revealed the presence of 8 glucosinolates, 12 anthocyanins, 2 carotenoids, and 7 phenylpropanoids. Glucosinolate contents varied among the different parts and types of kohlrabi. Glucoerucin contents were 4-fold higher in the flesh of purple kohlrabi than those in the skin. Among the 12 anthocyanins, cyanidin 3-(feruloyl)(sinapoyl) diglucoside-5-glucoside levels were the highest. Carotenoid levels were much higher in the skins than the flesh of both types of kohlrabi. The levels of most phenylpropanoids were higher in purple kohlrabi than in pale green ones. trans-Cinnamic acid content was 12.7-fold higher in the flesh of purple kohlrabi than that in the pale green ones. Thus, the amounts of glucosinolates, anthocyanins, carotenoids, and phenylpropanoids varied widely, and the variations in these compounds between the two types of kohlrabi were significant.