Simultaneous determination of 24 or more acidic and alkaline phytohormones in femtomole quantities of plant tissues by high-performance liquid chromatography–electrospray ionization–ion trap mass spectrometry

Phytohormones act at relatively low concentrations as major regulatory factors of plant growth and development, and cross talk of phytohormones is currently of great interest throughout the plant science community. To meet this demand, a method that is capable of simultaneously analyzing diverse plant hormones is essential. This paper introduces a high-performance liquid chromatographic separation technique coupled with sensitive and selective ion trap mass spectrometry to simultaneously determine 24 or more acidic and alkaline phytohormones, including auxin, cis- and trans-abscisic acid, 11 cytokinins, and 10 gibberellins, in a single injection of sample. A binary solid-phase extraction using Oasis MCX cartridges for cations and Oasis MAX cartridges for anions was used to prepurify more than 24 acidic and alkaline phytohormones from a single plant extract. The method showed good linearity for all 24 phytohormones with R ² values ranging from 0.9903 to 0.9997. Limits of detection for most of the phytohormones were in the femtomole range with some extending into the sub-femtomole range. This method was applied to hundreds of plant samples comprising different tissues from various plants, including herbaceous, woody climbing, and woody plants to demonstrate feasibility and to validate the methodology.     

Separation and quantification of four isomers of indole-3-acetyl-myo-inositol in plant tissues using high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry

Indole-3-acetyl-myo-inositol (IAInos) is one of the most important auxin conjugates for storage and transportation of auxin. The information of its composition, distribution, and metabolism is particularly desired for elucidating the related signal transduction pathways of the plant hormones. However, separation and quantification of the four individual IAInos isomers in plant tissues have not been reported so far. In this work, we first synthesized and isolated four IAInos isomers using semi-preparative high-performance liquid chromatography (HPLC). The IAInos isomer structures were characterized using liquid chromatography-electrospray ionization quadrupole time-of-flight tandem mass spectrometry (LC-QTOF/MS) and nuclear magnetic resonance spectroscopy (NMR). Using these pure compounds as internal or external standards, an efficient LC-MS method was developed for simultaneous detection of indole-3-acetic acid, methyl indole-3-acetic acid ester, and the four IAInos isomers in plant tissue samples. The linear working range and lower limit of detection for the four IAInos isomers are 10–2,000 ng mL^?1 and 5.0 ng mL^?1, respectively. The stabilities and interconversion pathways of IAInos isomers were studied using our synthetic isomers. It was found that two IAInos isomers existed in Zea mays kernels, while all of the four IAInos isomers were present in the roots of Arabidopsis thaliana. The content of IAInos in A. thaliana roots was much lower than in the Z. mays kernels. The methodology in this article provides useful techniques and methods for systematic study on the phytophysiology and phytochemistry of IAA conjugates and other related plant hormones.

The extraction,antioxidant and against β-amyloid induced toxicity of polyphenols from Alsophila spinulosa leaves

Alsophila spinulosa is a tree-like fern, and many evidences suggested that plant polyphenols had the potential therapeutic for Alzheimer s disease (AD). Herein, polyphenols (ASP) was isolated from A. spinulosa leaves and its major constituent were isoorientin and vitexin. ASP displayed excellent antioxidant activity and obvious anti-lipid peroxidation capacity in vitro. ASP improved the survival rate of C. elegans under high temperature by enhancing the antioxidant enzymes activities and decreasing the lipid peroxidation level. Moreover, ASP alleviated β-amyloid (Aβ) induced paralysis and reduced Aβ deposition, decreased reactive oxygen species (ROS) accumulation and improved the level of skn-1 mRNA. In addition, ASP decreased the levels of pdk-1 and akt-1 mRNA in P13K/AKT signaling pathway. In conclusion, ASP may be a potential ingredient for the alleviation of AD.     

AtTrxh3, a Thioredoxin,Is Identified as an Abscisic AcidBinding Protein in Arabidopsis thaliana

Abscisic acid (ABA, 1) is a plant hormone that regulates various plant physiological processes such as seed developing and stress responses. The ABA signaling system has been elucidated; binding of ABA with PYL proteins triggers ABA signaling. We have previously reported a new method to isolate a protein targeted with a bioactive small molecule using a biotin linker with alkyne and amino groups, a protein cross-linker, and a bioactive small molecule with an azido group (azido probe). This method was used to identify the unknown ABA binding protein of Arabidopsis thaliana. As a result, AtTrxh3, a thioredoxin, was isolated as an ABA binding protein. Our developed method can be applied to the identification of binding proteins of bioactive compounds.     

Fluorescence studies on nyctinasty using fluorescence labeled cis-p-coumaroylagmatine, a leaf-opening substance of Albizzia plants: existence of genus-specific receptor for leaf-movement factor

We developed fluorescent probes (1 and 2) based on the structure of cis-p-coumaroylagmatine (3), a leaf-opening substance of Albizzia julibrissin Durazz. These probes were effective for the leaf-opening of A. julibrissin, and specifically bind to the motor cell of this plant. Moreover, binding of the fluorescent probe was specific to the plant motor cell contained in the plants belonging to the Albizzia genus. These results showed that the binding of a probe compound with a motor cell is specific to the plant genus and suggested that the genus-specific receptor molecule for the leaf-movement factor on a motor cell would be involved in nyctinasty.     

Phylogenetic Analysis of Putative Genes Involved in the Tryptophan-Dependent Pathway of Auxin Biosynthesis in Rice

Plant proteome databases were mined for a flavin monooxygenase (YUCCA), tryptophan decarboxylase (TDC), nitrilase (NIT), and aldehyde oxidase (AO) enzymes that could be involved in the tryptophan-dependent pathway of auxin biosynthesis. Phylogenetic trees for enzyme sequences obtained were constructed. The YUCCA and TDC trees showed that these enzymes were conserved across the plant kingdom and therefore could be involved in auxin synthesis. YUCCAs branched into two clades. Most experimentally studied YUCCAs were found in the first clade. The second clade which has representatives from only seed plants contained Arabidopsis sequences linked to embryonic development. Therefore, sequences in this clade were suggested to be evolved with seed development. Examination of TDC activity and expression had previously linked this enzyme to secondary products synthesis. However, the phylogenetic finding of a conserved TDC clade across land plants suggested its essential role in plant growth. Phylogenetic analysis of AOs showed that plants inherited one AO. Recent gene duplication was suggested as AO sequences from each species were similar to each other rather than to AO from other species. Taken together and based on the experimental support of the involvement of AO in abscisic synthesis, AO was excluded as an intermediate in IAA production. Phylogenetic tree for NIT showed that the first clade contained sequences from species across the plant kingdom whereas the second branch contained sequences from only Brassicaceae. Even though NIT4 orthologues were conserved in the second clade, their major role seems to be detoxification of hydrogen cyanide rather than producing IAA.     

A Stable and Efficient Agrobacterium tumefaciens-Mediated Genetic Transformation of the Medicinal Plant Digitalis purpurea L.

In this study, we developed a rapid and efficient method for in vitro propagation and Agrobacterium tumefaciens-mediated transformation of Digitalis purpurea L. (syn. foxglove), an important medicinal plant. Mature leaf explants of D. purpurea were used for 100 % adventitious shoot regeneration on Murashige and Skoog (MS) medium supplemented with 1 mg L^?1 thidiazuron (TDZ) (a cytokine) and 0.1 mg L^?1 1-naphthaleneacetic acid (NAA) (an auxin). Transformation was achieved by inoculating leaf explants with the A. tumefaciens strains GV2260/pBI121 or GV3101/pBI121. The binary vector pBI121 contained the reporter β-glucuronidase gene (GUS) and kanamycin selection marker nptII. Kanamycin-resistant shoots were regenerated directly on the selection medium 4–6 weeks after co-cultivation. Approximately, 52.2 and 60 % of kanamycin-resistant shoots transformed with Agrobacterium strains GV2260 and GV3101, respectively, showed strong GUS staining by histochemical assay. Furthermore, PCR and Southern blot analysis confirmed the presence of nptII and GUS on the chromosome of the transformed D. purpurea plants, and stable GUS expression was detected in the transformants by RT-PCR analysis. This efficient method of shoot regeneration and genetic transformation of D. purpurea will provide a powerful tool to increase and produce valuable components such as digitoxin, digoxin, and digoxigenin in D. purpurea through improved secondary metabolic pathways via a biotechnological approach.     

Verbenanone,an octahydro-5H-chromen-5-one from a Hawaiian-plant associated fungus FT431

Verbenanone (1), a new secondary metabolite with a unique (4aS,8aS)-octahydro-5H-chromen-5-one moiety has been obtained from the endophytic fungus FT431, which was isolated from the native Hawaiian plant Verbena sp. The structure of compound 1 was characterized based on NMR and MS spectroscopic analysis. The absolute configuration (AC) of compound 1 was determined by Mosher acids. Compound 1 was tested against A2780 and A2780cisR, but it was inactive.     

Characterization,selenylation, and antineoplastic effects on HepG2 cells in vitro and in vivo of an arabinofuranan from the fruits of Akebia quinata

Akebia quinata is a traditional medicinal plant distributed in East Asia and its fruits are applicated in food and pharmaceutical fields. Herein, a novel polysaccharide (AQP70-2A) with a molecular weight of 1.49 × 10⁴ Da was isolated from the fruits of A. quinata. Results of the chemical and spectroscopic analysis indicated that AQP70-2A was an arabinofuranan with a backbone mainly consisting of → 5)-α-l-Araf-(1→, →3,5)-α-l-Araf-(1→, and → 2,3,5)-α-l-Araf-(1→, and it also contained two types of branch chains. At the cellular level, AQP70-2A did not show significant antitumor properties, while selenylation significantly made the inhibitory effect of this natural macromolecule on HepG2 cells to be increased. Furthermore, the zebrafish xenograft model confirmed that selenized polysaccharide Se-AQP70-2A effectively blocked hepatocellular carcinoma cells invasion and metastasis. Meanwhile, the inhibition of Se-AQP70-2A on development of intersegmental vessels revealed its antiangiogenic activity.     

Lignins Isolated via Catalyst-Free Organosolv Pulping from Miscanthus x giganteus,M. sinensis,M. robustus and M. nagara: A Comparative Study

As a low-input crop, Miscanthus offers numerous advantages that, in addition to agricultural applications, permits its exploitation for energy, fuel, and material production. Depending on the Miscanthus genotype, season, and harvest time as well as plant component (leaf versus stem), correlations between structure and properties of the corresponding isolated lignins differ. Here, a comparative study is presented between lignins isolated from M. x giganteus, M. sinensis, M. robustus and M. nagara using a catalyst-free organosolv pulping process. The lignins from different plant constituents are also compared regarding their similarities and differences regarding monolignol ratio and important linkages. Results showed that the plant genotype has the weakest influence on monolignol content and interunit linkages. In contrast, structural differences are more significant among lignins of different harvest time and/or season. Analyses were performed using fast and simple methods such as nuclear magnetic resonance (NMR) spectroscopy. Data was assigned to four different linkages (A: β-O-4 linkage, B: phenylcoumaran, C: resinol, D: β-unsaturated ester). In conclusion, A content is particularly high in leaf-derived lignins at just under 70% and significantly lower in stem and mixture lignins at around 60% and almost 65%. The second most common linkage pattern is D in all isolated lignins, the proportion of which is also strongly dependent on the crop portion. Both stem and mixture lignins, have a relatively high share of approximately 20% or more (maximum is M. sinensis Sin2 with over 30%). In the leaf-derived lignins, the proportions are significantly lower on average. Stem samples should be chosen if the highest possible lignin content is desired, specifically from the M. x giganteus genotype, which revealed lignin contents up to 27%. Due to the better frost resistance and higher stem stability, M. nagara offers some advantages compared to M. x giganteus. Miscanthus crops are shown to be very attractive lignocellulose feedstock (LCF) for second generation biorefineries and lignin generation in Europe.     

Zanthoxylum Species: A Comprehensive Review of Traditional Uses,Phytochemistry, Pharmacological and Nutraceutical Applications

Zanthoxylum species (Syn. Fagara species) of the Rutaceae family are widely used in many countries as food and in trado-medicinal practice due to their wide geographical distribution and medicinal properties. Peer reviewed journal articles and ethnobotanical records that reported the traditional knowledge, phytoconstituents, biological activities and toxicological profiles of Z. species with a focus on metabolic and neuronal health were reviewed. It was observed that many of the plant species are used as food ingredients and in treating inflammation, pain, hypertension and brain diseases. Over 500 compounds have been isolated from Z. species, and the biological activities of both the plant extracts and their phytoconstituents, including their mechanisms of action, are discussed. The phytochemicals responsible for the biological activities of some of the species are yet to be identified. Similarly, biological activities of some isolated compounds remain unknown. Taken together, the Z. species extracts and compounds possess promising biological activities and should be further explored as potential sources of new nutraceuticals and drugs.     

Evaluation of microbial loads,physical characteristics,chemical constituents and biological properties of radiation processed Fagonia arabica

Whole plant of Fagonia arabica with 3 different particle sizes (30, 50 and 70 mesh) were exposed to gamma radiation doses of 1–10 kGy from a Cobalt 60 source. A series of tests was performed in order to check the feasibility of irradiation processing of the plant. The applied radiation doses did not affect (P<0.05) pH and antimicrobial activities of the plant. The total weight of the dry extracts in methanol as well as water was found increased with irradiation. The irradiated samples showed significant increase in phenolic content and free radical scavenging activity using DPPH. Shortly after irradiation (on the day of radiation treatment) high amounts of free radicals were detected in the irradiated plant samples and the chemiluminescence measurements were generally found to be dose dependent. Maximum luminescence intensity was observed in case of samples with mesh size of 30 for all the radiation doses applied. After a period of one month the chemiluminescence signals of the irradiated samples approximated those of the controls. The study suggests that gamma irradiation treatment is effective for quality improvement and enhances certain beneficial biological properties of the treated materials.     

Cross-mixing study of a poisonous Cestrum species,Cestrum diurnum in herbal raw material by chemical fingerprinting using LC-ESI-QTOF-MS/MS

Poisonous plants are widely distributed and may have risk of phytotoxicity upon mixing with medicinal plants. Several species of Cestrum genus are poisonous and linked with many serious health issues. In the present study, cross-mixing of a toxic plant, Cestrum diurnum with morphologically resembling medicinal plant, Adhatoda vasica was studied using chemical fingerprinting approach. LC-ESI-MS/MS tool was used to develop the chemical fingerprints of three toxic species of Cestrum, including, C. diurnum, C. nocturnum and C. parqui. Total forty-three compounds were identified using high-resolution LC-ESI-MS/MS data comparison. Chemometric analyses were done to compare the distribution of identified compounds present in these Cestrum species. One of the identified compounds, nornicotine (a toxic compound) was also quantified using LC-IT-MS/MS. Adulteration study was conducted by mixing toxic C. diurnum in A. vasica with various ratios (w/w) and five differentiable compounds were identified to detect the adulteration. The method was able to detect up to the limit of 5% mixing of toxic C. diurnum. Moreover, cytotoxicity of the methanolic extracts of these three species were also studied on normal human PBMC (peripheral blood mononuclear cells) and all found to be toxic, while the C. nocturnum showed the highest level of toxicity with the IC₅₀ 12.5 μg/mL.     

A Multiwell-Based Assay for Screening Thyroid Hormone Signaling Disruptors Using thibz Expression as a Sensitive Endpoint in Xenopus laevis

There is a need for rapidly screening thyroid hormone (TH) signaling disruptors in vivo considering the essential role of TH signaling in vertebrates. We aimed to establish a rapid in vivo screening assay using Xenopus laevis based on the T3-induced Xenopus metamorphosis assay we established previously, as well as the Xenopus Eleutheroembryonic Thyroid Assay (XETA). Stage 48 tadpoles were treated with a series of concentrations of T3 in 6-well plates for 24 h and the expression of six TH-response genes was analyzed for choosing a proper T3 concentration. Next, bisphenol A (BPA) and tetrabromobisphenol A (TBBPA), two known TH signaling disruptors, were tested for determining the most sensitive TH-response gene, followed by the detection of several suspected TH signaling disruptors. We determined 1 nM as the induction concentration of T3 and thibz expression as the sensitive endpoint for detecting TH signaling disruptors given its highest response to T3, BPA, and TBBPA. And we identified betamipron as a TH signaling agonist, and 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) as a TH signaling antagonist. Overall, we developed a multiwell-based assay for rapidly screening TH signaling disruptors using thibz expression as a sensitive endpoint in X. laevis.     

Signaling in the Tomato Immunity against Fusarium oxysporum

New strategies of control need to be developed with the aim of economic and environmental sustainability in plant and crop protection. Metabolomics is an excellent platform for both understanding the complex plant–pathogen interactions and unraveling new chemical control strategies. GC-MS-based metabolomics, along with a phytohormone analysis of a compatible and incompatible interaction between tomato plants and Fusarium oxysporum f. sp. lycopersici, revealed the specific volatile chemical composition and the plant signals associated with them. The susceptible tomato plants were characterized by the over-emission of methyl- and ethyl-salicylate as well as some fatty acid derivatives, along with an activation of salicylic acid and abscisic acid signaling. In contrast, terpenoids, benzenoids, and 2-ethylhexanoic acid were differentially emitted by plants undergoing an incompatible interaction, together with the activation of the jasmonic acid (JA) pathway. In accordance with this response, a higher expression of several genes participating in the biosynthesis of these volatiles, such as MTS1, TomloxC, TomloxD, and AOS, as well as JAZ7, a JA marker gene, was found to be induced by the fungus in these resistant plants. The characterized metabolome of the immune tomato plants could lead to the development of new resistance inducers against Fusarium wilt treatment.     

The discovery of novel auxin transport inhibitors by molecular modeling and three-dimensional pattern analysis

Summary Molecular modeling techniques and three-dimensional (3D) pattern analysis have been used to investigate the chemical and steric properties of compounds that inhibit transport of the plant hormone auxin. These compounds bind to a specific site on the plant plasma membrane characterized by its affinity for the herbicide N-1-naphthylphthalamic acid (NPA). A 3D model was derived from critical features of a set of ligands for the NPA receptor, a suggested binding conformation is proposed, and implications for the topographical features of the NPA receptor are discussed. This model, along with 3D structural analysis techniques, was then used to search the Abbott corporate database of chemical structures. Of the 467 compounds that satisfied the criteria of the model, 77 representative molecules were evaluated for their ability to compete for the binding of [³H]NPA to corn microsomal membranes. Nineteen showed activity that ranged from 16 to 85% of the maximum NPA binding. Four of the most active of these, representing chemical classes not included in the original compound set, were also found to inhibit polar auxin transport through corn coleoptile sections. Thus, this study demonstrates that 3D analysis techniques can identify active, novel ligands for biochemical target sites with concomitant physiological activity.     

Network Pharmacology-Based Study to Uncover Potential Pharmacological Mechanisms of Korean Thistle (Cirsium japonicum var. maackii (Maxim.) Matsum.) Flower against Cancer

Cirsium japonicum var. maackii (Maxim.) Matsum. or Korean thistle flower is a herbal plant used to treat tumors in Korean folk remedies, but its essential bioactives and pharmacological mechanisms against cancer have remained unexplored. This study identified the main compounds(s) and mechanism(s) of the C. maackii flower against cancer via network pharmacology. The bioactives from the C. maackii flower were revealed by gas chromatography-mass spectrum (GC-MS), and SwissADME evaluated their physicochemical properties. Next, target(s) associated with the obtained bioactives or cancer-related targets were retrieved by public databases, and the Venn diagram selected the overlapping targets. The networks between overlapping targets and bioactives were visualized, constructed, and analyzed by RPackage. Finally, we implemented a molecular docking test (MDT) to explore key target(s) and compound(s) on AutoDockVina and LigPlot+. GC-MS detected a total of 34 bioactives and all were accepted by Lipinski’s rules and therefore classified as drug-like compounds (DLCs). A total of 597 bioactive-related targets and 4245 cancer-related targets were identified from public databases. The final 51 overlapping targets were selected between the bioactive targets network and cancer-related targets. With Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, a total of 20 signaling pathways were manifested, and a hub signaling pathway (PI3K-Akt signaling pathway), a key target (Akt1), and a key compound (Urs-12-en-24-oic acid, 3-oxo, methyl ester) were selected among the 20 signaling pathways via MDT. Overall, Urs-12-en-24-oic acid, 3-oxo, methyl ester from the C. maackii flower has potent anti-cancer efficacy by inactivating Akt1 on the PI3K-Akt signaling pathway.     

Secu'amamine A, a novel indolizidine alkaloid from Securinega suffruticosa var. amamiensis

A novel indolizidine alkaloid, secu'amamine A (1), was isolated from the leaves and twigs of the medicinal plant, Securinega suffruticosa var. amamiensis together with securinine (2). The structure and relative stereochemistry of 1 was elucidated by spectroscopic data and its absolute configuration was assigned on the basis of the OMe-madelate method.