Characterization of Endophytic Fungi, Acremonium sp., from Lilium davidii and Analysis of Its Antifungal and Plant Growth-Promoting Effects.

The present study was aimed at isolating endophytic fungi from the Asian culinary and medicinal plant Lilium davidii and analyzing its antifungal and plant growth-promoting effects. In this study, the fungal endophyte Acremonium sp. Ld-03 was isolated from the bulbs of L. davidii and identified through morphological and molecular analysis. The molecular and morphological analysis confirmed the endophytic fungal strain as Acremonium sp. Ld-03. Antifungal effects of Ld-03 were observed against Fusarium oxysporum, Botrytis cinerea, Botryosphaeria dothidea, and Fusarium fujikuroi. The highest growth inhibition, i.e., 78.39 ± 4.21%, was observed for B. dothidea followed by 56.68 ± 4.38%, 43.62 ± 3.81%, and 20.12 ± 2.45% for B. cinerea, F. fujikuroi, and F. oxysporum, respectively. Analysis of the ethyl acetate fraction through UHPLC-LTQ-IT-MS/MS revealed putative secondary metabolites which included xanthurenic acid, valyl aspartic acid, gancidin W, peptides, and cyclic dipeptides such as valylarginine, cyclo-[L-(4-hydroxy-Pro)-L-leu], cyclo(Pro-Phe), and (3S,6S)-3-benzyl-6-(4-hydroxybenzyl)piperazine-2,5-dione. Other metabolites included (S)-3-(4-hydroxyphenyl)-2-((S)-pyrrolidine-2-carboxamido)propanoic acid, dibutyl phthalate (DBP), 9-octadecenamide, D-erythro-C18-Sphingosine, N-palmitoyl sphinganine, and hydroxypalmitoyl sphinganine. The strain Ld-03 showed indole acetic acid (IAA) production with or without the application of exogenous tryptophan. The IAA ranged from 53.12 ± 3.20 µg ml-1 to 167.71 ± 7.12 µg ml-1 under different tryptophan concentrations. The strain was able to produce siderophore, and its production was significantly decreased with increasing Fe(III) citrate concentrations in the medium. The endophytic fungal strain also showed production of organic acids and phosphate solubilization activity. Plant growth-promoting effects of the strain were evaluated on in vitro seedling growth of Allium tuberosum. Application of 40% culture dilution resulted in a significant increase in root and shoot length, i.e., 24.03 ± 2.71 mm and 37.27 ± 1.86 mm, respectively, compared to nontreated control plants. The fungal endophyte Ld-03 demonstrated the potential of conferring disease resistance and plant growth promotion. Therefore, we conclude that the isolated Acremonium sp. Ld-03 should be further investigated before utilization as a biocontrol agent and plant growth stimulator.

Discovery of novel potential plant growth regulators from Corydalis mucronifera.

Three pairs of enantiomers mucroniferals A-C (1-3), with a novel skeleton of 1,4-epoxynaphthalene-2,3-dicarboxylic acid first reported from nature source, were isolated from Corydalis mucronifera. Their structures were elucidated based on extensive spectroscopic data analysis of MS, 1D and 2D NMR, and their absolute configurations were confirmed by single-crystal X-ray diffraction analysis and comparison of the experimental and calculated ECD data. Mucroniferals A-C showed broad-spectrum inhibitory activities on seedling growth of all plants tested (Lepidium apetalum, Raphanus sativus, Lactuca sativa, and Arabidopsis thaliana) with a dose-dependent relationship. Additionally, mucroniferals A and B exhibited significant inhibitory effects on germination of most seeds at concentration of 80 µg/mL, and the inhibition was reversible.

Production, extraction and characterization of Chlorella vulgaris soluble polysaccharides and their applications in AgNPs biosynthesis and biostimulation of plant growth.

Chlorella vulgaris, like a wide range of other microalgae, are able to grow mixotrophically. This maximizes its growth and production of polysaccharides (PS). The extracted polysaccharides have a complex monosaccharide composition (fructose, maltose, lactose and glucose), sulphate (210.65 ± 10.5 mg g-1 PS), uronic acids (171.97 ± 5.7 mg g-1 PS), total protein content (32.99 ± 2.1 mg g-1 PS), and total carbohydrate (495.44 ± 8.4 mg g-1 PS). Fourier Transform infrared spectroscopy (FT-IR) analysis of the extracted polysaccharides showed the presence of N-H, O-H, C-H, -CH3, >CH2, COO-1, S=O and the C=O functional groups. UV-Visible spectral analysis shows the presence of proteins, nucleic acids and chemical groups (ester, carbonyl, carboxyl and amine). Purified polysaccharides were light green in color and in a form of odorless powder. It was soluble in water but insoluble in other organic solvents. Thermogravimetric analysis demonstrates that Chlorella vulgaris soluble polysaccharide is thermostable until 240°C and degradation occurs in three distinct phases. Differential scanning calorimetry (DSC) analysis showed the characteristic exothermic transition of Chlorella vulgaris soluble polysaccharides with crystallization temperature peaks at 144.1°C, 162.3°C and 227.7°C. The X-ray diffractogram illustrated the semicrystalline nature of these polysaccharides. Silver nanoparticles (AgNPs) had been biosynthesized using a solution of Chlorella vulgaris soluble polysaccharides. The pale green color solution of soluble polysaccharides was turned brown when it was incubated for 24 hours with 100 mM silver nitrate in the dark, it showed peak maximum located at 430 nm. FT-IR analysis for the biosynthesized AgNPs reported the presence of carbonyl, -CH3, >CH2, C-H,-OH and -NH functional groups. Scanning and transmission electron microscopy show that AgNPs have spherical shape with an average particle size of 5.76. Energy-dispersive X-ray (EDX) analysis showed the dominance of silver. The biosynthesized silver nanoparticles were tested for its antimicrobial activity and have positive effects against Bacillus sp., Erwinia sp., Candida sp. Priming seeds of Triticum vulgare and Phaseolus vulgaris with polysaccharides solutions (3 and 5 mg mL-1) resulted in significant enhancement of seedling growth. Increased root length, leaf area, shoot length, photosynthetic pigments, protein content, carbohydrate content, fresh and dry biomass were observed, in addition these growth increments may be attributed to the increase of antioxidant activities.

Ionic liquid-organic-functionalized ordered mesoporous silica-integrated dispersive solid-phase extraction for determination of plant growth regulators in fresh Panax ginseng.

The massive accumulation of plant growth regulators (PGRs) in Panax ginseng causes serious harm to human health. A new analytical method for the simultaneous determination of multiple PGRs in 19 types of fresh Panax ginseng is developed by a new designed wool cluster-inspired ionic liquid-functionalized ordered mesoporous silica-integrated dispersive solid-phase extraction coupled to high performance liquid chromatography (IL-WFOMS-I-DSPE-HPLC). The proposed method combines the advantages of the multiple adsorption mechanisms, high mass transfer rate and large adsorption capacity of the synthesized IL-WFOMS adsorbent with the safe, convenient operation of the new designed I-DSPE method. Under optimized conditions, the recoveries at three spike levels were in a range of 77.6-98.3% for 3-indole acetic acid (IAA), 3-indole propionic acid (IPA), 3-indole butyric acid (IBA), and 1-naphthaleneacetic acid (NAA) with the relative standard deviations (RSD) ≤8.6%, n = 3. This method exhibits the advantages of safety, convenience, reliability, and has great potential for simultaneous determination of multiple trace PGRs in complex sample matrices.

Isolation and Purification of Potent Growth Inhibitors from Piper methysticum Root.

Piper methysticum (kava) root is known to possess promising weed suppressing activity. The present study was conducted to search for potent plant growth inhibitors from the root of this medicinal pepper plant. The ethyl acetate (EtOAc) extract exhibited the strongest reduction on growth of Raphanus sativus (radish) (IC50 shoot and root growth = 172.00 and 51.31 µg/mL respectively) among solvent extracts. From this active extract, nine potent growth inhibitors involved in the inhibitory activities of P. methysticum root were isolated, purified and characterized by column chromatography (CC), gas chromatography-mass spectrometry (GC-MS), electrospray ionization-mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR). The six fractions purified by CC included two flavanones: 5-hydroxy-4',7-dimethoxyflavanone (C1) and 5,7-dihydroxy-4'-methoxy-6,8-dimethylflavanone (matteucinol, C2) and six kavalactones: 5,6-dehydro-kavain (C3), a mixture of kavain and yagonin (C4), yagonin (C5) and dihydro-5,6-dehydrokavain, 7,8-dihydrokavain, dihydromethysticin and methysticin (C6). The amounts of 5-hydroxy-4',7-dimethoxyflavanone, matteucinol, 5,6-dehydrokavain and yangonin were 0.76, 2.50, 2.75 and 2.09 mg/g dry weight (DW), respectively. The two flavanones C1 and C2 exhibited the strongest inhibition on shoot elongation (IC50 = 120.22 and 248.03 µg/mL, respectively), whilst the two kavalactone mixtures C4 and C6 showed the highest suppression on root growth of R. sativus (IC50 = 7.70 and 15.67 µg/mL, respectively). This study was the first to report the purification and inhibitory activities of the two flavanones 5-hydroxy-4',7-dimethoxyflavanone and matteucinol in P. methysticum root. The isolated constituents from P. methysticum root including the flavanones C1 and C2 and the mixtures C4 and C6 may possess distinct modes of action on plant growth. Findings of this study highlighted that the combinations of hexane-ethyl acetate by 9:1 and 8:2 ratios successfully purified flavanones and kavalactones in P. methysticum root.

Immunoaffinity chromatography combined with tandem mass spectrometry: A new tool for the selective capture and analysis of brassinosteroid plant hormones.

Brassinosteroids (BRs) are plant-specific steroid hormones that play essential roles in the regulation of many important physiological processes in plant life. Their extremely low concentrations (~pmoles/g FW) in plant tissue and huge differences in polarity of individual members within the BR family hamper their detection and quantification. To address this problem, an immunoaffinity sorbent with broad specificity and high capacity for different BR metabolites containing a monoclonal antibody (mAb) against a BR spacer (20S)-2α,3α-dihydroxy-7-oxa-7α-homo-5α-pregnane-6-one-20 carboxylic acid (BR4812) was used for the rapid and highly selective isolation of endogenous BRs containing a 2α,3α-diol in ring A from minute plant samples. This enrichment procedure was successfully applied as a sample preparation method prior to quantitative analysis of BRs in real plant tissues by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Use of immunoaffinity chromatography (IAC) increased the sensitivity of the UHPLC-MS/MS analysis owing to improvements in the BR signal-to-noise ratio (S/N) and matrix factor (MF). Although MF values of BRs analyzed in classical samples ranged from 8.9% to 47.4%, MF values for the IAC purified samples reached 44.5-96.6%. Thus, the developed IAC-UHPLC-MS/MS approach was shown to be a simple, robust, effective and extremely fast procedure requiring minute amounts of plant samples suitable for the quantitative profiling of many BR metabolites, helping to overcome the major problems associated with their determination in very complex plant matrices.

Protocol for Extraction and Isolation of Brassinosteroids from Plant Tissues.

Brassinosteroids (BRs) are a class of steroid plant hormones that participate with other plant hormones in the regulation of numerous developmental processes, including root and shoot growth, vascular differentiation, fertility, and seed germination. A characteristic feature of all plant hormones, including BRs, is that their concentration is extremely low in plant tissues and, therefore, the methods dealing with their determination belong to ultra-trace analysis, for which very sensitive analytical tools are needed. The analysis of natural BRs is essential when their functions and roles in plant growth and development are to be elucidated. Here, we describe a reliable protocol for high-throughput extraction and purification of BRs. The procedure consists of two solid-phase extraction steps and provides selective enrichment and efficient cleanup of these compounds from complex plant extracts. The protocol is designed for sensitive liquid chromatography-tandem mass spectrometry-based method for simultaneous detection of 22 naturally occurring BRs, including their biosynthetic precursors and most of their biologically active metabolites, without need for derivatization.

Hormonal profiling: Development of a simple method to extract and quantify phytohormones in complex matrices by UHPLC-MS/MS.

Plant growth regulators (PGRs) are very different chemical compounds that play essential roles in plant development and the regulation of physiological processes. They exert their functions by a mechanism called cross-talk (involving either synergistic or antagonistic actions) thus; it is for great interest to study as many PGRs as possible to obtain accurate information about plant status. Much effort has been applied to develop methods capable of analyze large numbers of these compounds but frequently excluding some chemical families or important PGRs within each family. In addition, most of the methods are specially designed for matrices easy to work with. Therefore, we wanted to develop a method which achieved the requirements lacking in the literature and also being fast and reliable. Here we present a simple, fast and robust method for the extraction and quantification of 20 different PGRs using UHPLC-MS/MS optimized in complex matrices.

Reproduction of the Medicinal Plant Pelargonium sidoides via Somatic Embryogenesis.

The medicinal plant Pelargonium sidoides DC. (Geraniaceae) was traditionally used for the treatment of the common cold and cough in South Africa. Today an aequous-ethanolic root extract from this plant is approved for the treatment of acute bronchitis and is globally marketed also as an immunostimulant. The increasing demand of the plant material for the industrial production indicates the need of new effective methods for the propagation of P. sidoides. Here we report somatic embryogenesis and in vitro plantlet regeneration from somatic cells of inflorescence shoots and petioles of P. sidoides. A one-week cultivation of explants in media containing different concentrations of thidiazuron (1, 2.2, 3, and 4 mg/L) followed by a cultivation period without phytohormones resulted in the induction of somatic embryos within 2-4 weeks. After 2-4 months, the embryos generated roots and could be transferred into a greenhouse, where flower formation took place and the development of seeds occurred with high germination rates. The root umckalin concentration, determined by high-performance thin-layer chromatography, was comparable to that of seed-cultivated plants (100 ± 6 vs. 113 ± 10 µg umckalin/g dried roots). For the first time, direct somatic embryogenesis has been established as an appropriate cultivation method for P. sidoides plants used as raw material in the pharmaceutical industry. Moreover, genetically identical plants (chemical races) can be easily generated by this procedure.

Accurate Analysis and Evaluation of Acidic Plant Growth Regulators in Transgenic and Nontransgenic Edible Oils with Facile Microwave-Assisted Extraction-Derivatization.

Determination of plant growth regulators (PGRs) in a signal transduction system (STS) is significant for transgenic food safety, but may be challenged by poor accuracy and analyte instability. In this work, a microwave-assisted extraction-derivatization (MAED) method is developed for six acidic PGRs in oil samples, allowing an efficient (<1.5 h) and facile (one step) pretreatment. Accuracies are greatly improved, particularly for gibberellin A3 (-2.72 to -0.65%) as compared with those reported (-22 to -2%). Excellent selectivity and quite low detection limits (0.37-1.36 ng mL(-1)) are enabled by fluorescence detection-mass spectrum monitoring. Results show the significant differences in acidic PGRs between transgenic and nontransgenic oils, particularly 1-naphthaleneacetic acid (1-NAA), implying the PGRs induced variations of components and genes. This study provides, for the first time, an accurate and efficient determination for labile PGRs involved in STS and a promising concept for objectively evaluating the safety of transgenic foods.

An ultrahigh-performance liquid chromatography method with electrospray ionization tandem mass spectrometry for simultaneous quantification of five phytohormones in medicinal plant Glycyrrhiza uralensis under abscisic acid stress.

An efficient simplified method was developed to determine multiple classes of phytohormones simultaneously in the medicinal plant Glycyrrhiza uralensis. Ultrahigh-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS) with multiple reaction monitoring (MRM) in negative mode was used for quantification. The five studied phytohormones are gibberellic acid (GA3), abscisic acid (ABA), jasmonic acid (JA), indole-3-acetic acid, and salicylic acid (SA). Only 100 mg of fresh leaves was needed, with one purification step based on C18 solid-phase extraction. Cinnamic acid was chosen as the internal standard instead of isotope-labeled internal standards. Under the optimized conditions, the five phytohormones with internal standard were separated within 4 min, with good linearities and high sensitivity. The validated method was applied to monitor the spatial and temporal changes of the five phytohormones in G. uralensis under ABA stress. The levels of GA3, ABA, JA, and SA in leaves of G. uralensis were increased at different times and with different tendencies in the reported stress mode. These changes in phytohormone levels are discussed in the context of a possible feedback regulation mechanism. Understanding this mechanism will provide a good chance of revealing the mutual interplay between different biosynthetic routes, which could further help elucidate the mechanisms of effective composition accumulation in medicinal plants.

[SYNTHESIS OF PHYTOHORMONES BY NOCARDIA VACCINII IMV B-7405--PRODUCER OF SURFACTANTS].

AIM: To study the synthesis of phytohormones (auxins, cytokinins, abscisic acid) under cultivation of Nocardia vaccinii IMV B-7405 (surfactants producer) in media containing different carbon sources (glycerol, refined sunflower oil, as well as waste oil after frying potatoes and meat). METHODS: Phytohormones were extracted from supernatants of culture liquid (before or after surfactant separation) by ethylacetate (auxins, abscisic acid) and n-butanol (cytokinins), concentrated and purified by thin-layer chromatography, then quantitative determination was performed using a scanning Sorbfil spectrodensitometer. RESULTS: While growing in medium with refined oil IMV B-7405 strain synthesized 1.8 ± 0.09 g/l extracellular surfactant, also maximum amount of auxins (245-770 µ/l) and cytokinins (134-348 µl). Cultivation of N. vaccini LMV B-7405 on waste oil was accompanied by decreasing amount of phytohormones to 23-84 µ/l (auxins) and 16-90 µ/l (cytokinins) and increasing surfactant concentration to 2.3-2.6 g/l. The level of abscisic acid synthesis was practically not dependent on the nature of growth substrate, was substantially lower than that of auxins and cytokinins and ranged from 2 to 12 µ/l. CONCLUSIONS: Obtained data demonstrate the possibility of using oil-containing industrial waste for the simultaneous synthesis of both surfactants and phytohormones, and indicate the need for studies of the effect of producer cultivation conditions on the biological properties of the target products of microbial synthesis.

Staurosporine from the endophytic Streptomyces sp. strain CNS-42 acts as a potential biocontrol agent and growth elicitor in cucumber.

Chinese medicinal plants and their surrounding rhizospheric soil serve as promising sources of actinobacteria. A total of 180 actinobacteria strains were isolated from the rhizosphere soil, leaves, stems, and roots of nine selected plants and have been identified as potential biocontrol agents against Fusarium oxysporum f. sp. cucumerinum. An endophytic strain CNS-42 isolated from Alisma orientale showed the largest zone of inhibition demonstrating a potent effect against F. oxysporum f. sp. cucumerinum and a broad antimicrobial activity against bacteria, yeasts, and other pathogenic fungi. The in vivo biocontrol assays showed that the disease severity index was significantly reduced (P < 0.05), and plant shoot fresh weight and height increased greatly (P < 0.05) in plantlets treated with strain CNS-42 compared to the negative control. This isolate was identified as Streptomyces sp. based on cultural, physiological, morphological characteristics, and 16S rRNA gene analysis. Further bioassay-guided isolation and purification revealed that staurosporine was responsible for its antifungal and plant growth promoting activities and the latter property of staurosporine is reported for the first time. The in vivo assay was further performed and indicated that staurosporine showed good growth promoting effect on the plant shoot biomass of cucumber. This is the first critical evidence identifying CNS-42 as a biocontrol agent for the soil borne pathogen, F. oxysporum f. sp. cucumerinum.

Development of a rapid LC-DAD/FLD method for the simultaneous determination of auxins and abscisic acid in plant extracts.

Plant hormones play a crucial role in controlling plant growth and development. These groups of naturally occurring substances trigger physiological processes at very low concentrations, which mandate sensitive techniques for their quantitation. This paper describes a method to quantify endogenous (±)-2-cis-4-trans-abscisic acid, indole-3-acetic acid, indole-3-propionic acid, and indole-3-butyric acid. The method combines high-performance liquid chromatography (HPLC) with diode array and fluorescence detection in a single run. Hybrid tea rose 'Monferrato' matrices (leaves, petals, roots, seeds, androecium, gynoecium, and pollen) were used as references. Rose samples were separated and suspended in extracting methanol, after which (±)-2-cis-4-trans-abscisic acid and auxins were extracted by solvent extraction. Sample solutions were added first to cation solid phase extraction (SPE) cartridges and the eluates to anion SPE cartridges. The acidic hormones were bound to the last column and eluted with 5% phosphoric acid in methanol. Experimental results showed that this approach can be successfully applied to real samples and that sample preparation and total time for routine analysis can be greatly reduced.

Harzianolide, a novel plant growth regulator and systemic resistance elicitor from Trichoderma harzianum.

A detailed understanding of the effect of natural products on plant growth and protection will underpin new product development for plant production. The isolation and characterization of a known secondary metabolite named harzianolide from Trichoderma harzianum strain SQR-T037 were described, and the bioactivity of the purified compound as well as the crude metabolite extract in plant growth promotion and systemic resistance induction was investigated in this study. The results showed that harzianolide significantly promoted tomato seedling growth by up to 2.5-fold (dry weight) at a concentration of 0.1 ppm compared with the control. The result of root scan suggested that Trichoderma secondary metabolites may influence the early stages of plant growth through better root development for the enhancement of root length and tips. Both of the purified harzianolide and crude metabolite extract increased the activity of some defense-related enzymes to response to oxidative stress. Examination of six defense-related gene expression by real-time reverse transcription-PCR analysis revealed that harzianolide induces the expression of genes involved in the salicylic acid (PR1 and GLU) and jasmonate/ethylene (JERF3) signaling pathways while crude metabolite extract inhibited some gene expression (CHI-II and PGIP) related to basal defense in tomato plants. Further experiment showed that a subsequent challenge of harzianolide-pretreated plants with the pathogen Sclerotinia sclerotiorum resulted in higher systemic resistance by the reduction of lesion size. These results indicate that secondary metabolites of Trichoderma spp., like harzianolide, may play a novel role in both plant growth regulation and plant defense responses.

Protective effect of Polygonum orientale L. extracts against Clavibater michiganense subsp. sepedonicum, the causal agent of bacterial ring rot of potato.

The Polygonum orientale L. extracts were investigated for antibacterial activity against Clavibater michiganense subsp. sepedonicum (Spieckermann & Kotthoff) Davis et al., the causal agent of a serious disease called bacterial ring rot of potato. The results showed that the leaf extracts of P. orientale had significantly (p<0.05) greater antibacterial activity against C. michiganense subsp. sepedonicum than root, stem, flower extracts in vitro. According to the results of single factor experiments and L(27)3(13) orthogonal experiments, optimum extraction conditions were A1B3C1, extraction time 6 h, temperature 80°C, solid to liquid ratio 1∶10 (g:mL). The highest (p<0.05) antibacterial activity was observed when pH was 5, excluding the effect of control. The extracts were stable under ultraviolet (UV). In vivo analysis revealed that 50 mg/mL of P. orientale leaf extracts was effective in controlling decay. Under field conditions, 50 mg/mL of P. orientale leaf extracts also improved growth parameters (whole plant length, shoot length, root length, plant fresh weight, shoot fresh weight, root fresh weight, dry weight, and number of leaves), in the 2010 and 2011 two growing seasons. Further solvent partition assays showed that the most active compounds were in the petroleum ether fractionation. Transmission electron microscopy (TEM) showed drastic ultrastructural changes caused by petroleum ether fractionation, including bacterial deformation, electron-dense particles, formation of vacuoles and lack of cytoplasmic materials. These results indicated that P. orientale extracts have strong antibacterial activity against C. michiganense subsp. sepedonicum and a promising effect in control of bacterial ring rot of potato disease.

Orally active hypoglycemic protein from Costus igneus N. E. Br.: an in vitro and in vivo study.

Plants have been used for the treatment of diabetes since time immemorial. In the present study, insulin-like protein (ILP) is purified from Costus igneus belonging to family Costaceae from Western ghats of India. The ILP showed cross reactivity with murine anti-insulin antibodies hence was purified by affinity chromatography using anti-insulin antibodies. The characterization of ILP showed that it is structurally different from insulin but functionally similar. The ILP showed a hypoglycemic activity in an in vitro assay with insulin responsive cell line RIN 5f. Interestingly ILP showed significant decrease in blood glucose level when administered orally in oral glucose tolerance test. This was compared to insulin a positive control given intraperitoneally in streptozotocine induced diabetic mice. There was no toxic effect seen on animals after administrating the ILP. Therefore we conclude that the ILP purified in the present study from C. igneus is a novel protein having hypoglycemic activity.

Synthesis of inositol phosphate ligands of plant hormone-receptor complexes: pathways of inositol hexakisphosphate turnover.

Reduction of phytate is a major goal of plant breeding programs to improve the nutritional quality of crops. Remarkably, except for the storage organs of crops such as barley, maize and soybean, we know little of the stereoisomeric composition of inositol phosphates in plant tissues. To investigate the metabolic origins of higher inositol phosphates in photosynthetic tissues, we have radiolabelled leaf tissue of Solanum tuberosum with myo-[2-3H]inositol, undertaken a detailed analysis of inositol phosphate stereoisomerism and permeabilized mesophyll protoplasts in media containing inositol phosphates. We describe the inositol phosphate composition of leaf tissue and identify pathways of inositol phosphate metabolism that we reveal to be common to other kingdoms. Our results identify the metabolic origins of a number of higher inositol phosphates including ones that are precursors of cofactors, or cofactors of plant hormone-receptor complexes. The present study affords alternative explanations of the effects of disruption of inositol phosphate metabolism reported in other species, and identifies different inositol phosphates from that described in photosynthetic tissue of the monocot Spirodela polyrhiza. We define the pathways of inositol hexakisphosphate turnover and shed light on the occurrence of a number of inositol phosphates identified in animals, for which metabolic origins have not been defined.

Identification of two phytotoxins, blumenol A and grasshopper ketone, in the allelopathic Japanese rice variety Awaakamai.

Aqueous methanol extracts of the traditional rice (Oryza sativa) variety Awaakamai, which is known to have the greatest allelopathic activity among Japanese traditional rice varieties, inhibited the growth of roots and shoots of cress (Lepidium sativum), lettuce (Lactuca sativa), timothy (Phleum pratense), Digitaria sanguinalis, Lolium multiflorum and Echinochloa crus-galli. Increasing the extract concentration increased the inhibition, suggesting that the extract of Awaakamai contains growth inhibitory substances. The extract of Awaakamai was purified and two main growth inhibitory substances were isolated and determined by spectral data as blumenol A and grasshopper ketone. Blumenol A and grasshopper ketone, respectively, inhibited the growth of cress shoots and roots at concentrations greater than 10 and 30 µmol/L. The concentrations required for 50% growth inhibition on cress roots and shoots were 84 and 27 µmol/L, respectively, for blumenol A, and 185 and 76 µmol/L, respectively, for grasshopper ketone. These results suggest that blumenol A and grasshopper ketone may contribute to the growth inhibitory effect of Awaakamai and may play an important role in the allelopathy of Awaakamai.

A liquid chromatography tandem mass spectrometry method for simultaneous determination of acid/alkaline phytohormones in grapes.

A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneous determination of five acid/alkaline phytohormones, i.e., indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthylacetic acid (NAA), gibberellic acid (GA(3)) and isopentenyladenine (2IP), in grapes was developed. After optimization, the samples were extracted with methanol containing 1% formic acid and purified by Oasis HLB SPE cartridges. The analytes were separated on a Thermo Hypersil Gold column (100 mm×2.1 mm, 3.0 µm) with water and acetonitrile, then determined with Thermo tandem quadrupole mass spectrometer operating in negative electro-spray ionization using selected reaction monitoring (SRM) mode. The established method was further validated by determining the linearity (R² ≥ 0.9990), average recovery (82.5-105.4%), sensitivity (0.05-1.00 ng mL⁻¹), precision (RSD ≤1 3.0%) and stability (RSD ≥ 82.0%). Finally, the application of the approach proposed to thirty grape samples convinced its desirable performance for rapid analysis of multiclass phytohormones, supporting its sufficient capability for multiresidue analyses or other analytical system targeting phytohormones in agriculture field.