Induced transcriptional profiling of phenylpropanoid pathway genes increased flavonoid and lignin content in Arabidopsis leaves in response to microbial products.

BACKGROUND: The production and use of biologically derived soil additives is one of the fastest growing sectors of the fertilizer industry. These products have been shown to improve crop yields while at the same time reducing fertilizer inputs to and nutrient loss from cropland. The mechanisms driving the changes in primary productivity and soil processes are poorly understood and little is known about changes in secondary productivity associated with the use of microbial products. Here we investigate secondary metabolic responses to a biologically derived soil additive by monitoring changes in the phenlypropanoid (PP) pathway in Arabidopsis thaliana. RESULTS: This study was designed to test the influence of one of these products (Soil Builder™-AF, SB) on secondary metabolism after being applied at different times. One time (TI) application of SB to Arabidopsis increased the accumulation of flavonoids compared to multiple (TII) applications of the same products. Fourteen phenolic compounds including flavonols and anothocyanins were identified by mass spectrometry. Kaempferol-3,7-O-bis-α-L-rhamnoside and quercetin 3,7-dirhamnoside, the major compounds, increased 3-fold and 4-fold, respectively compared to control in the TI treatment. The most abundant anthocyanin was cyanidin 3-rhamnoglucoside, which increased 3-fold and 2-fold in TI compared to the control and TII, respectively. Simultaneously, the expression of genes coding for key enzymes in the PP pathway (phenylalanine ammonia lyase, cinnamate 4-hydroxylase, chalcone synthase, flavonoid-3'-O-hydroxylase, flavonol synthase1 and dihydroflavonol-4-reductase) and regulatory genes (production of anthocyanin pigment2, MYB12, MYB113, MYB114, EGL3, and TT8) were up-regulated in both treatments (TI and TII). Furthermore, application of TI and TII induced expression of the lignin pathway genes (hydroxyl cinamyl transferase, caffeyl-CoA O-methyl transferase, cinnamyl alcohol dehydrogenase, cinnamyl-CoA reductase, secondary wall-associated NAC domain protein1, MYB58 and MYB63 resulting in higher accumulation of lignin content compared to the control. CONCLUSIONS: These results indicate that the additions of microbially based soil additives have a perceptible influence on phenylpropanoid pathway gene regulation and its production of secondary metabolites. These findings open an avenue of research to investigate the mode of action of microbially-based soil additives which may assist in the sustainable production of food, feed, fuel and fiber.

A functional EDS1 ortholog is differentially regulated in powdery mildew resistant and susceptible grapevines and complements an Arabidopsis eds1 mutant.

Vitis vinifera (grapevine) is the most economically important deciduous fruit crop, but cultivated grapevine varieties lack adequate innate immunity to a range of devastating diseases. To identify genetic resources for grapevine innate immunity and understand pathogen defense pathways in a woody perennial plant, we focus in this study on orthologs of the central Arabidopsis thaliana defense regulator ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1). The family of EDS1-like genes is expanded in grapevine, and members of this family were previously found to be constitutively upregulated in the resistant variety 'Norton' of the North American grapevine species Vitis aestivalis, while they were induced by Erysiphe necator, the causal agent of grapevine powdery mildew (PM), in the susceptible V. vinifera variety 'Cabernet Sauvignon'. Here, we determine the responsiveness of individual EDS1-like genes in grapevine to PM and salicylic acid, and find that EDS1-like paralogs are differentially regulated in 'Cabernet Sauvignon', while two are constitutively upregulated in 'Norton'. Sequencing of VvEDS1 and VaEDS1 cDNA and genomic clones revealed high conservation in the protein-encoding sequence and some divergence of the promoter sequence in the two grapevine varieties. Complementation of the Arabidopsis eds1-1 mutant showed that the EDS1-like gene with highest predicted amino acid sequence similarity to AtEDS1 from either grapevine varieties is a functional ortholog of AtEDS1. Together, our analyses show that differential susceptibility to PM is correlated with differences in EDS1 expression, not differences in EDS1 function, between resistant 'Norton' and susceptible 'Cabernet Sauvignon'.

Methyl jasmonate and salicylic acid induced oxidative stress and accumulation of phenolics in Panax ginseng bioreactor root suspension cultures.

To investigate the enzyme variations responsible for the synthesis of phenolics, 40 day-old adventitious roots of Panax ginseng were treated with 200 microM methyl jasmonate (MJ) or salicylic acid (SA) in a 5 L bioreactor suspension culture (working volume 4 L). Both treatments caused an increase in the carbonyl and hydrogen peroxide (H2O2) contents, although the levels were lower in SA treated roots. Total phenolic, flavonoid, ascorbic acid, non-protein thiol (NPSH) and cysteine contents and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical reducing activity were increased by MJ and SA. Fresh weight (FW) and dry weight (DW) decreased significantly after 9 days of exposure to SA and MJ. The highest total phenolics (62%), DPPH activity (40%), flavonoids (88%), ascorbic acid (55%), NPSH (33%), and cysteine (62%) contents compared to control were obtained after 9 days in SA treated roots. The activities of glucose 6-phosphate dehydrogenase, phenylalanine ammonia lyase, substrate specific peroxidases (caffeic acid peroxidase, quercetin peroxidase and ferulic acid peroxidase) were higher in MJ treated roots than the SA treated ones. Increased shikimate dehydrogenase, chlorogenic acid peroxidase and beta-glucosidase activities and proline content were observed in SA treated roots than in MJ ones. Cinnamyl alcohol dehydrogenase activity remained unaffected by both MJ and SA. These results strongly indicate that MJ and SA induce the accumulation of phenolic compounds in ginseng root by altering the phenolic synthesis enzymes.

Copper-induced changes in the growth, oxidative metabolism, and saponin production in suspension culture roots of Panax ginseng in bioreactors.

Roots of Panax ginseng exposed to various concentrations of Cu (0.0, 5, 10.0, 25.0, and 50.0 microM) accumulated high amounts of Cu in a concentration-dependent and duration-dependent manner. Roots treated with 50 microM Cu resulted in 52% and 89% growth inhibition after 20 and 40 days, respectively. Saponin synthesis was stimulated at a Cu concentration between 5 and 25 muM but decreased at 50 microM Cu. Malondialdehyde content (MDA), lipoxygenase activity (LOX), superoxide ion (O2*-) accumulation, and H2O2 content at 5 and 10 microM Cu-treated roots were not increased but strongly increased at 50 microM Cu resulting in the oxidation of ascorbate (ASC) and glutathione (GSH) to dehydroascorbate (DHA) and glutathione disulfide (GSSG), respectively indicating a clear oxidative stress. Seven well-resolved bands of superoxide dismutase (SOD) were detected in the gel and an increase in SOD activity seemed to be mainly due to the induction of Fe-SOD 3. Five to 10 microM Cu slightly induced activity of ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR), guaiacol peroxidase (G-POD) but inhibited monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR) enzyme activities. No changes in catalase (CAT) activity and in activity gel were found up to 25 microM Cu, but both G-POD and CAT activities were inhibited at 50 microM Cu. Glutathione metabolism enzymes such as gamma-glutamylcysteine synthetase (gamma-GCS), glutathione-S-transferase (GST), and glutathione peroxidase activities (GPx) were activated at 5 and 10 microM Cu but were strongly inhibited at 50 microM Cu due to the Cu accumulation in root tissues. The strong depletion of GSH at 50 microM Cu was associated to the strong induction of gamma-glutamyltranspeptidase (gamma-GGT) activity. These results indicate that plant could grow under Cu stress (5-25 microM) by modulating the antioxidant defense mechanism for combating Cu induced oxidative stress.

Methyl jasmonate and salicylic acid elicitation induces ginsenosides accumulation, enzymatic and non-enzymatic antioxidant in suspension culture Panax ginseng roots in bioreactors.

The effects of methyl jasmonate (MJ) and salicylic acid (SA) on changes of the activities of major antioxidant enzymes, superoxide anion accumulation (O2-), ascorbate, total glutathione (TG), malondialdehyde (MDA) content and ginsenoside accumulation were investigated in ginseng roots (Panax ginseng L.) in 4 l (working volume) air lift bioreactors. Single treatment of 200 microM MJ and SA to P. ginseng roots enhanced ginsenoside accumulation compared to the control and harvested 3, 5, 7 and 9 days after treatment. MJ and SA treatment induced an oxidative stress in P. ginseng roots, as shown by an increase in lipid peroxidation due to rise in O2- accumulation. Activity of superoxide dismutase (SOD) was inhibited in MJ-treated roots, while the activities of monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), SOD, guaiacol peroxidase (G-POD), glutathione peroxidase (GPx) and glutathione reductase (GR) were induced in SA-treated roots. A strong decrease in the activity of catalase (CAT) was obtained in both MJ- and SA-treated roots. Activities of ascorbate peroxidase (APX) and glutathione S transferase (GST) were higher in MJ than SA while the contents of reduced ascorbate (ASC), redox state (ASC/(ASC+DHA)) and TG were higher in SA- than MJ-treated roots while oxidized ascorbate (DHA) decreased in both cases. The result of these analyses suggests that roots are better protected against the O2- stress, thus mitigating MJ and SA stress. The information obtained in this work is useful for efficient large-scale production of ginsenoside by plant-root cultures.

CO(2)-induced total phenolics in suspension cultures of Panax ginseng C. A. Mayer roots: role of antioxidants and enzymes.

The effects of different concentrations of CO(2) (1%, 2.5% and 5%) on the antioxidant capacity, total phenols, flavonoids, protein content and phenol biosynthetic enzymes in roots of Panax ginseng were studied in bioreactor (working volume 4 l) after 15, 30 and 45 days. CO(2) induced accumulation of total phenolics in a concentration and duration dependent manner. Total phenols, flavonoids and 1,1-diphenyl-2-picrylhydrazyl (DPPH) activity increased 60%, 30% and 20% at 2.5% CO(2) after 45 days compared to control in P. ginseng roots which indicated that phenolics compounds played an important role in protecting the plants from CO(2). Hypothesizing that increasing the phenolic compounds in roots of P. ginseng may increase its nutritional functionality; we investigated whether pentose phosphate pathway (PPP), shikimate/phenylpropanoid pathway enzymes have a role in phenolics mobilization in P. ginseng roots. Fresh weight (FW), dry weight (DW) and growth ratio was increased at 1% and 2.5% CO(2) only after 45 days, however, unaffected after 15 and 30 days. Results also indicated that high CO(2) progressively stimulated the activities of glucose 6 phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49), shikimate dehydrogenase (SKDH, E.C. 1.1.1.25), phenylalanine ammonia lyase (PAL, E.C. 4.3.1.5), cinnamyl alcohol dehydrogenase (CAD, E.C. 1.1.1.195), caffeic acid (CA) peroxidase and chlorogenic acid (CGA) peroxidase after 15, 30 and 45 days. Increased CO(2) levels resulted in increases in accumulation of total protein (45%), non-protein thiol (NP-SH) (30%) and cysteine contents (52%) after 45 days compared to control and increased activities of beta-glucosidase (GS, E.C. 3.2.1.21) and polyphenol oxidase (PPO, E.C. 1.10.3.2) in P. ginseng roots indicated that they played an important role in protecting the plants from CO(2). These results strongly suggest that high concentration of CO(2) delivered to ginseng root suspension cultures induced the accumulation of total phenolics possessing high antioxidant properties probably useful for human health. Therefore, roots of P. ginseng are considered as a good source of phenolics compounds with high antioxidants capacity and can be produced on a large scale.

Effects of temperature on oxidative stress defense systems, lipid peroxidation and lipoxygenase activity in Phalaenopsis.

Higher plants growing in natural environments experience various abiotic stresses. The aim of this study was to determine whether exposure to temperature-stress would lead to oxidative stress and whether this effect varied with different exposure periods. The thermal dependencies of the activities of protective enzymes, photosynthetic efficiency (Fv/Fm), protein, non-protein thiol (NP-SH), cysteine content, lipoxygenase (LOX) activity (EC 1.13.11.12) and malondialdehyde (MDA) content at 25-40 degrees C were determined for 4, 24 and 48 h in leaf and root segments of Phalaenopsis. The increase in MDA level and LOX activity may be due to temperature-associated oxidative damage to leaf and root segments. Temperature-stress induced not only activities of active oxygen species (AOS) scavenging enzymes but also protein, NP-SH and cysteine content in both leaf and root segments at 30 degrees C for 4 and 24 h (except for 48 h in some cases) compared to 25 degrees C-and greenhouse-grown leaf and root segments indicating that antioxidants enzymes played an important role in protecting plant from temperature-stress. However, activities of dehydroascorbate reductase (DHAR, EC 1.8.5.1), glutathione peroxidase (GPX, EC 1.11.1.9) and glutathione-S-transferase (GST, EC 2.5.1.18) in leaf and root, glutathione reductase (GR, EC 1.6.4.2) in leaf and guaiacol peroxidase (G-POD, 1.11.1.7) in root segments were induced significantly at 40 degrees C compared to 25 degrees C and greenhouse-grown plants suggesting that these enzymes play protective roles at high temperature. In contrast, activities of superoxide dismutase (SOD, EC 1.15.1.1) and monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) in leaf and root, catalase (CAT, EC 1.11.1.6) in root, GR in root, and protein, cysteine, NP-SH content in both root and leaf and Fv/Fm ratio were diminished significantly at 40 degrees C compared to 25 degrees C-and greenhouse-grown plants. These indicate that these enzymes were apparently not involved in detoxification process and sensitive at higher temperature. Also, the close relation between activities of enzymes with their metabolites at 30 degrees C than 40 degrees C indicated that the antioxidants enzymes and metabolites both may play an important role in protecting cells against the temperature-stress.