Enhancement of Macarpine Production in Eschscholzia Californica Suspension Cultures under Salicylic Acid Elicitation and Precursor Supplementation.

Macarpine is a minor benzophenanthridine alkaloid with interesting biological activities, which is produced in only a few species of the Papaveraceae family, including Eschscholzia californica. Our present study was focused on the enhancement of macarpine production in E. californica suspension cultures using three elicitation models: salicylic acid (SA) (4; 6; 8 mg/L) elicitation, and simultaneous or sequential combinations of SA and L-tyrosine (1 mmol/L). Sanguinarine production was assessed along with macarpine formation in elicited suspension cultures. Alkaloid production was evaluated after 24, 48 and 72 h of elicitation. Among the tested elicitation models, the SA (4 mg/L), supported by L-tyrosine, stimulated sanguinarine and macarpine production the most efficiently. While sequential treatment led to a peak accumulation of sanguinarine at 24 h and macarpine at 48 h, simultaneous treatment resulted in maximum sanguinarine accumulation at 48 h and macarpine at 72 h. The effect of SA elicitation and precursor supplementation was evaluated also based on the gene expression of 4'-OMT, CYP719A2, and CYP719A3. The gene expression of investigated enzymes was increased at all used elicitation models and their changes correlated with sanguinarine but not macarpine accumulation.

A Fluorescence Anisotropy-Based Comprehensive Method for the In Vitro Screening of COI1-JAZs Agonists and Antagonists.

The phytohormone (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile) causes protein-protein interactions (PPI) between F-box Protein CORONATINE INSENSITIVE 1 (COI1) and JASMONATE ZIM DOMAIN (JAZ) transcriptional repressor. A total of 13 JAZ subtypes are encoded in the genome of Arabidopsis thaliana; however, their genetic redundancy obfuscates the individual function of each JAZ. One approach to decipher this redundant signaling network is chemical genetics, using small molecules specific to individual JAZ subtype, for which a reliable and high-throughput screening system of the ligands for all combinations of COI1-JAZs would be indispensable. In this chapter, we describe a fluorescence anisotropy-based quantitative screening system for the ligands of COI1-JAZ co-receptors. Our method is applicable to agonists and antagonists of the COI1-JAZs.

Chitosan and chitosan nanoparticles induced expression of pathogenesis-related proteins genes enhances biotic stress tolerance in tomato.

The aim of this work was to evaluate the possibility of control of wilt disease caused by Fusarium andiyazi through chitosan (CS) and chitosan nanoparticles (CNPs). In the present study, the expression pattern of pathogenesis-related (PR) proteins genes such as PR-1, PR-2 (ß-1,3-glucanase), PR-8 (chitinase), and PR-10 was analyzed using real-time RT-PCR. In vitro studies showed that among different concentrations (0.1-5.0 mg/ml), 5.0 mg/ml concentration of CS and CNPs produced maximum inhibition of radial mycelial growth, 54.8% and 73.81%, respectively. Also, upregulated expression of ß-1,3-glucanase, chitinase, PR-1 and PR-10 genes were recorded with 1.48, 1.15, 1.15, and 1.41, fold expression in 24 hpi, respectively, in plants inoculated with CNPs. The most significant up-regulation was observed in transcript profile of SOD that showed 4.5-foldexpression, at 48 hpi. Therefore, our results confirmed that CS and CNPs induced up-regulation of PR-proteins and antioxidant genes might play a significant role for successful biocontrol.

Peroxidase from jackfruit: Purification, characterization and thermal inactivation.

Peroxidase (POD) from jackfruit bulb was purified using ammonium sulfate precipitation, hydrophobic interaction and gel filtration columns. The POD was a dimer with a molecular weight of 104kDa. The Km and Vmax values for guaiacol, gallic acid and o­phenylenediamine (OPD) were estimated. OPD was the most suitable substrate. The enzyme showed its maximum activity at pH5.5 and 55-60°C. The activation energy (Ea) of heat inactivation was estimated to be 206.40kJ/mol. The enthalpy, free energy and entropy values for the thermal inactivation were also determined. The POD activity was enhanced by K+, Zn2+, Ba2+, citric acid, malic acid, benzoic acid and EDTA·Na2, but inhibited by Cu2+, Ca2+, glutathione, cysteine and ascorbic acid. Chemical modification indicated a histidine residue was located in the enzyme active site. The POD activity in fruit extracts significantly decreased when heated at 80°C and 90°C. The ferric-reducing antioxidant power, ABTS radical scavenging activity and total phenolics decreased with increasing heating temperature and time.

Aluminium-induced cell death requires upregulation of NtVPE1 gene coding vacuolar processing enzyme in tobacco (Nicotiana tabacum L.).

Cell death mechanism triggered by aluminium (Al) ion was investigated at root apex of tobacco (cultivar Bright Yellow) and in cultured tobacco cell line BY-2 derived from Bright Yellow, focusing on VPE genes (NtVPE1a, NtVPE1b, NtVPE2, NtVPE3). Cell death was detected as a loss of integrity of the plasma membrane by vital staining with fluorescein diacetate (in root apex) and Evans blue (in BY-2), respectively. At root apex, the upregulation of gene expression of VPE1a and VPE1b was observed significantly after 9h of Al exposure in parallel with an enhancement of cell death, while the upregulation of VPE2 and VPE3 were observed later. Similarly, in BY-2 cells, the upregulation of VPE1a and VPE1b and the enhancement of cell death were synchronously observed after 3-h exposure to Al, while the upregulation of VPE2 and VPE3 occurred later. RNA interference (RNAi) lines of each of the VPEs were constructed in BY-2 cells. Comparative studies between wild-type and the RNAi lines indicated that both Al-enhanced VPE activity and Al-induced cell death were significantly suppressed in the RNAi lines of VPE1 (dual suppressor of VPE1a and VPE1b), but not in the RNAi lines of VPE2 and that of VPE3. Taken together, we conclude that the upregulation of VPE1 gene expression and following enhancement of VPE activity under Al stress cause cell death in actively growing or elongating cells of tobacco.

Relationship between aluminum stress and caffeine biosynthesis in suspension cells of Coffea arabica L.

Toxicity by aluminum is a growth-limiting factor in plants cultivated in acidic soils. This metal also promotes signal transduction pathways leading to the biosynthesis of defense compounds, including secondary metabolites. In this study, we observed that Coffea arabica L. cells that were kept in the dark did not produce detectable levels of caffeine. However, irradiation with light and supplementation of the culture medium with theobromine were the best conditions for cell maintenance to investigate the role of aluminum in caffeine biosynthesis. The addition of theobromine to the cells did not cause any changes to cell growth and was useful for the bioconversion of theobromine to caffeine. During a short-term AlCl3-treatment (500µM) of C. arabica cells kept under light irradiation, increases in the caffeine levels in samples that were recovered from both the cells and culture media were evident. This augmentation coincided with increases in the enzyme activity of caffeine synthase (CS) and the transcript level of the gene encoding this enzyme (CS). Together, these results suggest that actions by Al and theobromine on the same pathway lead to the induction of caffeine biosynthesis.

Helminthosporic acid functions as an agonist for gibberellin receptor.

Helminthosporol was isolated from a fungus, Helminthosporium sativum, as a natural plant growth regulator in 1963. It showed gibberellin-like bioactivity that stimulated the growth of the second leaf sheath of rice. After studying the structure-activity relationship between the compound and some synthesized analogs, it was found that helminthosporic acid (H-acid) has higher gibberellin-like activity and chemical stability than helminthosporol. In this study, we showed that (1) H-acid displays gibberellin-like activities not only in rice but also in Arabidopsis, (2) it regulates the expression of gibberellin-related genes, (3) it induces DELLA degradation through binding with a gibberellin receptor (GID1), and (4) it forms the GID1-(H-acid)-DELLA complex to transduce the gibberellin signal in the same manner as gibberellin. This work shows that the H-acid mode of action acts as an agonist for gibberellin receptor.

1-N-histidine phosphorylation of ChlD by the AAA+ ChlI2 stimulates magnesium chelatase activity in chlorophyll synthesis.

Magnesium chelatase (Mg-chelatase) inserts magnesium into protoporphyrin during the biosynthesis of chlorophyll and bacteriochlorophyll. Enzyme activity is reconstituted by forming two separate preactivated complexes consisting of a GUN4/ChlH/protoporphyrin IX substrate complex and a ChlI/ChlD enzyme 'motor' complex. Formation of the ChlI/ChlD complex in both Chlamydomonas reinhardtii and Oryza sativa is accompanied by phosphorylation of ChlD by ChlI, but the orthologous protein complex from Rhodobacter capsulatus, BchI/BchD, gives no detectable phosphorylation of BchD. Phosphorylation produces a 1-N-phospho-histidine within ChlD. Proteomic analysis indicates that phosphorylation occurs at a conserved His residue in the C-terminal integrin I domain of ChlD. Comparative analysis of the ChlD phosphorylation with enzyme activities of various ChlI/ChlD complexes correlates the phosphorylation by ChlI2 with stimulation of Mg-chelatase activity. Mutation of the H641 of CrChlD to E641 prevents both phosphorylation and stimulation of Mg-chelatase activity, confirming that phosphorylation at H641 stimulates Mg-chelatase. The properties of ChlI2 compared with ChlI1 of Chlamydomonas and with ChlI of Oryza, shows that ChlI2 has a regulatory role in Chlamydomonas.

Phenotypic and biochemical alterations in relation to MT2 gene expression in Plantago ovata Forsk under zinc stress.

Plantago ovata Forsk is an annual herb with immense medicinal importance, the seed and husk of which is used in the treatment of chronic constipation, irritable bowel syndrome, diarrhea since ancient times. Zinc, an essential metal, is required by plants as they form important components of zinc finger proteins and also aid in synthesis of photosynthetic pigments such as chlorophyll. However, in excess amount Zn causes chlorosis of leaf and shoot tissues and generate reactive oxygen species. The present study is aimed at investigating the changes in expression levels of MT2 gene in Plantago ovata under zinc stress. Data show up to 1.66 fold increase in expression of PoMT2 in 1000 µM ZnSO4·7H2O treated sample. Our study also describes alteration of MT2 gene expressions in Plantago ovata as observed through Real time PCR (qPCR) done by [Formula: see text] method. In this study we have observed an upregulation (or induction) in the PoMT2 gene expression level in 500 and 800 µM ZnSO4·7H2O treated samples but found saturation on further increasing the dose to 1000 µM of ZnSO4·7H2O. Determination of the phenotypic and biochemical changes in Plantago ovata due to exposure to zinc stress of concentrations 500, 800 and 1000 µM revealed oxidative stress. The enhanced expression of MT2 gene in Plantago ovata has a correlation with the increased total antioxidant activity and increased DPPH radical scavenging activity.

Low molecular weight chitosan is an effective antifungal agent against Botryosphaeria sp. and preservative agent for pear (Pyrus) fruits.

Antifungal activity and preservative effect of a low molecular weight chitosan (LMWC) sample, derived from chitosan by enzymatic hydrolysis, were investigated in vitro and in vivo. A pathogenic fungal strain was isolated from decayed pear (Pyrus bretschneideri cv. "Huangguan") fruit and identified as Botryosphaeria sp. W-01. LMWC was shown to strongly inhibit W-01 growth based on studies of minimum inhibitory concentration (MIC) and effects on mycelial biomass and radial growth of the fungus. LMWC treatment of W-01 cells reduced ergosterol synthesis and mitochondrial membrane potential (ΔY), early events of apoptosis. Transmission electron microscopy and confocal laser scanning microscopy studies revealed that LMWC penetrated inside W-01 hyphae, thereby inducing ultrastructural damage. LMWC coating had a significant preservative effect on wounded and nonwounded pear fruits, by inhibiting postharvest decay and browning processes. LMWC activated several defense-related enzymes (polyphenol oxidase, peroxidase, chitinase), maintained nutritional value, and slowed down weight loss. Our findings indicate the strong potential of LMWC as a natural preservative agent for fruits and vegetables.

The role of sirtuins in cellular homeostasis.

Sirtuins are evolutionarily conserved nicotinamide adenine dinucleotide (NAD(+))-dependent lysine deacylases or ADP-ribosyltransferases. These cellular enzymes are metabolic sensors sensitive to NAD(+) levels that maintain physiological homeostasis in the animal and plant cells.

Elicitation and treatment with precursors of phenolics synthesis improve low-molecular antioxidants and antioxidant capacity of buckwheat sprouts.

BACKGROUND: Recently, an increase of interest in the modification of food products on each step of production (breeding, production technology, storage condition) is observed. Nutritional properties as well as level and activity of bioactive compounds in plant-origin food may be modified using a range of technological and biotechnological practices and elicitation should be mentioned between them. METHODS: Elicitation with willow bark infusion supported by feeding with the phenylpropanoid pathway precursors were used for improving the quality of buckwheat sprouts. Special emphasis has been placed on the metabolomic and biochemical changes and the mechanism of overproduction of low-molecular antioxidants. RESULTS: The accumulation of phenolics is caused by stimulation of two main enzymes the phenylpropanoid pathway (tyrosine ammonia-lyase and phenylalanine ammonia-lyase). Tyrosine ammonia-lyase activities were effectively induced by feeding with tyrosine (about four times that of the control), whereas phenylalanine ammonia-lyase activity was the highest in the elicited control sprouts and those fed with shikimic acid (an increase by 60% compared to the control). Shikimic acid feeding (both elicited and non-elicited sprouts) effectively improved the total phenolics (by about 10% and 20%, respectively), condensed tannins (by about 30% and 28%, respectively), and flavonoids (by about 46% and 70%, respectively). Significant increase of vitexin, rutin, chlorogenic acid and isoorientin contents was also observed. The treatments increased the ascorbic acid content, too. Total antioxidant capacity of sprouts was most effectively increased by feeding with shikimic acid and further elicitation. CONCLUSIONS: The studies transfer biotechnology commonly used for the induction of overproduction of secondary metabolites in plant cell line systems to low-processed food production. The obtained results could be used for better understanding of the effect of elicitation and precursor feeding on antioxidants production and contribute to improving the buckwheat sprouts quality.

The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA.

[Effect of Azospirillum lectins on the Activity of Proteolytic Enzymes and Their Inhibitors in Wheat Seedling Roots].

The lectins of associative nitrogen-fixing strains Azospirillum brasilense Sp7 and Sp245 were shown to exerte a multidirectional effect on the activity of acidic (pH 3.5), neutral (6.8), and alkaline (pH 7.8) proteinases. The lectin of the epiphytic A. brasilense Sp7 decreased proteolytic activity at all pH values, whereas the lectin of the endophytic A. brasilense Sp245 activated neutral and alkaline proteinases, while not affecting the alkaline ones. Experiments with protease inhibitors made it possible to conclude that the lectins of the studied A. brasilense strains alter the ratio between the activities of different protease types in germinating seeds. The activity of trypsin inhibitors in wheat seedling roots was found to increase in the presence of the lectins. Our results indicate a broader spectrum of effects of azospirilla lectins on the host plant organism.

Short-term UV-B dose stimulates production of protective metabolites in Matricaria chamomilla leaves.

Physiological response of two cultivars of Matricaria chamomilla plants on UV irradiation was studied. The impact of used short-time UV dose was evaluated in three time points; 2, 24 and 48 h after irradiation. Used UV irradiation immediately resulted in changes in plant oxidative status monitored as increased concentration of H2 O2 . Decrease in chlorophyll a and b indicated the impact on photosynthetic apparatus. For phenolic secondary metabolites, an increase in total soluble phenols and AlCl3 -reactive flavonols was observed. The activity of main phenolic enzyme, phenylalanine ammonia-lyase, increased with time after irradiation. Significant changes, mainly decreasing trends, in the content of free coumarins and their glycosidic precursors were observed. Enhanced accumulation in chlorogenic and 1,5-dicaffeoylquinic acid and in (Z)-isoform of dicycloethers was detected. From these results, the redirecting precursors of coumarin biosynthesis to biosynthesis of substances with higher antioxidative potential can be assumed. Different reactions in diploid and tetraploid plants were recorded, too.

Eugenol enhances the resistance of tomato against tomato yellow leaf curl virus.

BACKGROUND: Tomato yellow leaf curl virus disease (TYLCVD) causes severe to economic losses in tomato crops in China. The control of TYLCVD is based primarily on the use of synthetic insecticide to control its vector whitefly (Bemisia tabaci). To look for an alternative method for disease control, we investigated the effect of eugenol on controlling TYLCVD. The potential of eugenol to trigger systemic acquired resistance (SAR) in tomato (Jiangsu 14) plants against TYLCV was also investigated. RESULTS: In greenhouse experiments, eugenol significantly reduced disease severity when applied as a foliar spray, thus demonstrating a systemic effect. The disease spread rapidly in control plants and by the end of the experiment almost all control plants showed severe symptoms. Eugenol also induced H2O2 accumulation in tomato plants. Activities of peroxidase (POD), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) were significantly induced compared with those of control plants. As further consequences, increase of salicylic acid (SA) levels and expression of PR-1 proteins, a molecular marker of SAR in tomato, could also be observed. CONCLUSION: This is the first report of eugenol as an elicitor and its ability to suppress plant virus diseases under greenhouse conditions. It is suggested that eugenol has the potential to be an effective biocontrol agent against TYLCV in tomato plants.

[Effect of adaptogenic preparations on Na+/H+-antiporter function in plasma membrane of corn root cells under salinity conditions].

Salinity is a hard stress factor for plant organisms which negative effect is caused chiefly by sodium toxic for plants. Plant cells try to remove Na+ from their cytoplasm outside and to vacuolar space by secondary active Na+/H+-antiporters. Their functions can be intensified by gene engineering methods however we try do it with the help of non-toxic bioactive preparations. A comparison of their effect on the plasma membrane of Na+/H+-antiporters was carried out on corn seedling roots of Zea mays L. exposed at 0.1 M NaCl. Before we have established that Methyure used by seed pretreating possesses a high salt protective ability as against Ivine. It was found that without NaCl exposition Na+/H+-antiporter activity in root plasma membrane was nearly unnoticeable but increased slightly with seedling age. Methyure and Ivine did not influence its activity in control root seedling. One day 0.1 M NaCl exposition evoked a considerable increasing of Na+/H+-antiporter activity and its gene expression but these effects disappeared at 10 day NaCl exposition. Methyure use reinforced Na+/H+-antiporter activity and prolonged it at NaCl exposition without effect on its gene expression whereas Ivine effects on these indexes were insignificant. Obtained results showed that the salt protective capability of Methyure is connected with plasma membrane Na+/H+-antiporter activation which is realized on molecular level.

Insights into the TOR-S6K signaling pathway in maize (Zea mays L.). pathway activation by effector-receptor interaction.

The primordial TOR pathway, known to control growth and cell proliferation, has still not been fully described for plants. Nevertheless, in maize, an insulin-like growth factor (ZmIGF) peptide has been reported to stimulate this pathway. This research provides further insight into the TOR pathway in maize, using a biochemical approach in cultures of fast-growing (FG) and slow-growing (SG) calli, as a model system. Our results revealed that addition of either ZmIGF or insulin to SG calli stimulated DNA synthesis and increased the growth rate through cell proliferation and increased the rate of ribosomal protein (RP) synthesis by the selective mobilization of RP mRNAs into polysomes. Furthermore, analysis of the phosphorylation status of the main TOR and S6K kinases from the TOR pathway revealed stimulation by ZmIGF or insulin, whereas rapamycin inhibited its activation. Remarkably, a putative maize insulin-like receptor was recognized by a human insulin receptor antibody, as demonstrated by immunoprecipitation from membrane protein extracts of maize callus. Furthermore, competition experiments between ZmIGF and insulin for the receptor site on maize protoplasts suggested structural recognition of the putative receptor by either effector. These data were confirmed by confocal immunolocalization within the cell membrane of callus cells. Taken together, these data indicate that cell growth and cell proliferation in maize depend on the activation of the TOR-S6K pathway through the interaction of an insulin-like growth factor and its receptor. This evidence suggests that higher plants as well as metazoans have conserved this biochemical pathway to regulate their growth, supporting the conclusion that it is a highly evolved conserved pathway.

[Influence of methanol on the content of NAD(P)H, free amino acids and protein in the cells of Chlamydomonas reinhartdii].

It is known that the addition of methanol to the culture medium stimulates the photosynthetic productivity of some species of microalgae, but its influence on the biochemical composition of the biomass has not been investigated. The aim of the present work is to determine the effect of methanol (50 mM) on the content of free amino acids, soluble proteins and reduced nicotinamide coenzyme NAD(P)H in C. reinhartdii cells. It is shown that in case of illumination of C. reinhardtii methanol raises intracellular content of NAD(P)H four times more efficiently than in the darkness. Total content of free amino acids is increased and their ratio is changed. The concentration of glutamic acid, glutamine, alanine, serine and tyrosine also increases. The concentration of valine and methionine is reduced. Growth of culture with methanol is followed by an increase in the content of intracellular protein by 30% after 20 hours of cultivation. The obtained data indicate that methanol stimulates growth of C. reinhardtii, not only as a result of additional carbon utilization, but also due to improved nitrogen assimilation and the impact on the energy metabolism of cells.

Key role of Fe(2+) in epithiospecifier protein activity.

The chemical nature of the hydrolysis products from the glucosinolate-myrosinase system depends on the presence or absence of supplementary proteins such as epithiospecifier proteins (ESPs). ESPs promote the formation of epithionitriles from terminal alkenyl glucosinolates and, as recent evidence suggests, simple nitriles at the expense of isothiocyanates. From a human health perspective isothiocyanates are the most important because they are major inducers of carcinogen-detoxifying enzymes. Fe(2+) is an essential factor in ESP activity, although several recent studies have highlighted discrepancies in the understanding of the ESP-iron interaction. To investigate further the role iron species play in regulating ESP activity, four ESP-containing seedpowders were analyzed for ESP and myrosinase activities, endogenous iron content, and glucosinolate degradation products after the addition of iron species, specific chelators, and reducing agents. For the first time this paper shows the effect of these additions on the hydrolysis of individual glucosinolates that constitute the total pool. Aged seeds and 3-day seedlings were also tested to investigate the effects of seed storage and early plant development on iron levels and ESP activity. The four ESP-containing plant systems tested gave two distinctive responses, thus providing strong evidence that ESPs vary markedly in their Fe(2+) requirement for activity. The results also indicated that reduction of ferric to ferrous iron drives variations in ESP activity during early plant development. The reverse oxidation reaction provided a convincing explanation for the loss of ESP activity during seed storage. Aged seeds produced seedlings with substantially lower ESP activity, and there was a concomitant loss in germination rate. It was concluded that manipulation of endogenous iron levels of ESP-containing plants could increase the conversion of glucosinolates to isothiocyanates and enhance potential health benefits.