A rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry method for determination of phytohormones in the medicinal plant saffron.

Saffron (Crocus sativus L.) is a valuable Chinese herb with high medicinal value. Saffron pistils are used as medicine, so increasing the number of flowers can increase the yield. Plant hormones have essential roles in the growth and development of saffron, as well as the response to biotic and abiotic stresses (especially in floral initiation), which may directly affect the number of flowers. Quantitative analysis of plant hormones provides a basis for more efficient research on their synthesis, transportation, metabolism, and action. However, starch (which interferes with extraction) is present in high levels, and hormone levels are extremely low, in saffron corms, thereby hampering accurate determination of plant-hormone levels in saffron. Herein, we screened an efficient and convenient pre-treatment method for plant materials containing abundant amounts of starch. Also, we proposed an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the quantification of abscisic acid (ABA) and auxin (IAA). Then, the method was applied for the detection of hormone-content differences between flowering and non-flowering top buds, as well as between lateral and top buds. Our method showed high sensitivity, reproducibility, and reliability. Specifically, good linearity in the range 2-100 ng ml-1 was achieved in the determination of ABA and IAA, and the correlation coefficient (R2) was >0.9982. The relative standard deviation was 2.956-14.51% (intraday) and 9.57-18.99% (interday), and the recovery range was 89.04-101.1% (n = 9). The matrix effect was 80.38-90.50% (n = 3). The method was thoroughly assessed employing various "green" chemistry evaluation tools: Blue Applicability Grade Index (BAGI), Complementary Green Analytical Procedure Index (Complex GAPI) and Red Green Blue 12 Algorithm (RGB12). These tools revealed the good greenness, analytical performance, applicability, and overall sustainability alignment of our method. Quantitative results showed that, compared with saffron with a flowering phenotype cultivated at 25 °C, the contents of IAA and ABA in the terminal buds of saffron cultivated at 16 °C decreased significantly. When cultivated at 25 °C, the IAA and ABA contents in the terminal buds of saffron were 1.54- and 4.84-times higher than those in the lateral buds, respectively. A simple, rapid, and accurate UPLC-MS/MS method was established to determine IAA and ABA contents. Using this method, a connection between the contents of IAA and ABA and the flowering phenotype was observed in the quantification results. Our data lay a foundation for studying the flowering mechanism of saffron.

Mitigation of the effect of drought on growth and yield of pomegranates by foliar spraying of different sizes of selenium nanoparticles.

BACKGROUND: Drought is a very important environmental stressor, which has negative effects on the growth of trees, decreasing their yield. The role of different-sized selenium nanoparticles (Se-NPs) in the mitigation of environmental stresses such as drought in crops has not yet been investigated. RESULTS: Trees treated with Se-NPs displayed higher levels of photosynthetic pigments, a better nutrient status, better physical parameters (especially fruit cracking) and chemical parameters, a higher phenolic content, and higher concentrations of osmolytes, antioxidant enzymes, and abscisic acid than untreated trees under drought stress. Foliar spraying of 10 and 50 nm Se-NPs alleviated many of the deleterious effects of drought in pomegranate leaves and fruits and this was achieved by reducing stress-induced lipid peroxidation and H2 O2 content by enhancing the activity of antioxidant enzymes. Furthermore, the 10 nm Se-NPs treatment produced more noticeable effects than the treatment with 50 nm Se-NPs. CONCLUSION: Results confirm the positive effects of nanoparticle spraying, especially the role of 10 nm Se-NPs in the management of negative effects of drought not only for pomegranates but potentially also for other crops. © 2021 Society of Chemical Industry.

Endogenous levels of cytokinins, indole-3-acetic acid and abscisic acid in in vitro grown potato: A contribution to potato hormonomics.

A number of scientific reports published to date contain data on endogenous levels of various phytohormones in potato (Solanum tuberosum L.) but a complete cytokinin profile of potato tissues, that would include data on all particular molecular forms of cytokinin, has still been missing. In this work, endogenous levels of all analytically detectable isoprenoid cytokinins, as well as the auxin indole-3-acetic acid (IAA), and abscisic acid (ABA) have been determined in shoots and roots of 30 day old in vitro grown potato (cv. Désirée). The results presented here are generally similar to other data reported for in vitro grown potato plants, whereas greenhouse-grown plants typically contain lower levels of ABA, possibly indicating that in vitro grown potato is exposed to chronic stress. Cytokinin N-glucosides, particularly N7-glucosides, are the dominant cytokinin forms in both shoots and roots of potato, whereas nucleobases, as the bioactive forms of cytokinins, comprise a low proportion of cytokinin levels in tissues of potato. Differences in phytohormone composition between shoots and roots of potato suggest specific patterns of transport and/or differences in tissue-specific metabolism of plant hormones. These results represent a contribution to understanding the hormonomics of potato, a crop species of extraordinary economic importance.

Abscisic acid identification in Okra, Abelmoschus esculentus L. (Moench): perspective nutraceutical use for the treatment of diabetes.

Okra, Abelmoschus esculentus L. (Moench), also known as Lady's Fingers, gombo, or bamje, is an annual plant belonging to the Malvaceae family. Traditional olistic medicine since centuries directly associates this plant and its parts to a beneficial health hypoglycemic effect. Since the abscisic acid (ABA) has been associated to an interesting hypoglycemic effect, this triggered us to verify and quantify the presence of the abscisic acid in the okra phytocomplex. In particular, ABA, a plant derived hormone, has been proven by recent studies to be effective on mammals. To determine and quantify the ABA content, different parts of the Okra plant extracts have been evaluated, and HPLC-DAD analysis has been used allowing us to report for the first time the presence of this isoprenoid compound. Bioaccessibility has been also investigated using a simulated gastro intestinal (GI) digestion protocol with the aim of explore the possibility of okra extract as nutraceutical.

Alginate oligosaccharide postharvest treatment preserve fruit quality and increase storage life via Abscisic acid signaling in strawberry.

Abscisic acid (ABA) has been advocated to play substantial role on ripening of non-climacteric fruit. Here we report that alginate oligosaccharide (AOS) postharvest treatments delayed the accumulation of ABA and ABA-conjugates and restrained the expression of ABA signaling genes, resulting enlarged storage life of strawberry. In addition, AOS postharvest treatments also increased the quality and reduced the degradation of cell wall components and repressed the expression of cell wall degradation genes. AOS treated fruits exhibited significant delays of hardness, decay percentage, titratable acidity, pH, total soluble solids and vitamin C content compared to untreated fruits. Moreover, AOS had a positive effect on retaining higher amount of anthocyanin, total phenol and flavonoids contents. The finding of this study suggests that AOS postharvest treatments are very useful for preserving fruit quality and enhancing shelf life by delayed ABA accretion, restrained the gene expression related to ABA signaling and cell wall degeneration.

Endorsing and extending the repertory of nutraceutical and antioxidant sources in mangoes during postharvest shelf life.

Mango byproducts, such as peels, contain high levels of antioxidants and fiber and represent important sources of nutraceuticals and pharmacological products. Fruit are collected at the mature green stage then stored and ripened, undergoing several structural and molecular changes over the course of this process. However, very little is known regarding the content and nature of antioxidant compounds in peels of elite and local cultivars during postharvest shelf life (PSL). We screened the phenolic compound content of six cultivars during PSL, including elite (Kent, Tommy, and Ataulfo) and local (Manila, Manililla, and Criollo) mangoes, using a targeted metabolomics approach. We determined that Ataulfo mangoes exhibited the highest content of phenolic compounds during PSL. Untargeted metabolomics and comparative proteomics in Ataulfo and Manililla showed these cultivars to be significant sources of phenolic and lipidic compounds, with the latter cultivar also representing an interesting candidate as a new source for nutraceutical products.

Simultaneous Determination of Abscisic Acid and Its Catabolites by Hydrophilic Solid-Phase Extraction Combined with Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry.

An efficient and selective pretreatment method of one-step hydrophilic interaction chromatography-based solid phase extraction (HILIC SPE) was developed using silica as the sorbent to quickly and sensitively detect endogenous ABA and its five catabolites in fresh Oryza sativa tissues. The extracted analytes were sensitively quantified with ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Under the optimized conditions, good linearity of the developed analytical method was obtained in the range of 0.2-1000 ng/mL with linear correlation coefficients ( r) greater than 0.9987. The limits of detection (LODs, signal/noise = 3) ranged from 0.01 to 0.74 ng/mL. The relative recoveries were between 83.3% and 112.0% with the relative standard deviations (RSDs) ranging from 0.5 to 15.0%. Using the proposed method, the concentration variations of ABA and its catabolites were monitored in the salt-stressed rice tissues.

Development of an ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method for the simultaneous determination of salicylic acid, jasmonic acid, and abscisic acid in rose leaves.

This paper describes a method to detect and quantitate the endogenous plant hormones (±)-2-cis-4-trans-abscisic acid, (-)-jasmonic acid, and salicylic acid by means of ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in hybrid rose leaf matrices. Deuterium-labeled [(2)H6] (+)-2-cis-4-trans-abscisic acid, [(2)H6] (±)-jasmonic acid, and [(2)H4]-salicylic acid were used as internal standards. Rose samples (10 mg) were extracted with methanol/water/acetic acid (10:89:1) and subsequently purified on an Oasis MCX 1 cm(3) Vac SPE cartridge. Performance characteristics were validated according to Commission Decision 2002/657/EC. Recovery, repeatability, and within-laboratory reproducibility were acceptable for all phytohormones tested at three different concentrations. The decision limit and detection capability for (±)-2-cis-4-trans-abscisic acid, (-)-jasmonic acid, and salicylic acid were 0.0075 and 0.015 µg/g, 0.00015 and 0.00030 µg/g, and 0.0089 and 0.018 µg/g, respectively. Matrix effects (signal suppression or enhancement) appeared to be high for all substances considered, implying the need for quantitation based on matrix-matched calibration curves.

Effects of nitric oxide treatment on the cell wall softening related enzymes and several hormones of papaya fruit during storage.

Papaya fruits (Carica papaya L. cv 'Sui you 2') harvested with < 5% yellow surface at the blossom end were fumigated with 60 microL/L of nitric oxide for 3 h and then stored at 20 degrees C with 85% relative humility for 20 days. The effects of nitric oxide treatment on ethylene production rate, the activities of cell wall softening related enzymes including polygalacturonase, pectin methyl esterase, pectate lyase and cellulase and the levels of hormones including indole acetic acid, abscisic acid, gibberellin and zeatin riboside were examined. The results showed that papaya fruits treated with nitric oxide had a significantly lower rate of ethylene production and a lesser loss of firmness during storage. A decrease in polygalacturonase, pectin methyl esterase, pectate lyase and cellulase activities was observed in nitric oxide treated fruit. In addition, the contents of indole acetic acid, abscisic acid and zeatin riboside were reduced in nitric oxide treated fruit, but no significant reduction in the level of gibberellin was found. These results indicate that nitric oxide treatment can effectively delay the softening and ripening of papaya fruit, likely via the regulation of cell wall softening related enzymes and certain hormones.

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.

Quantification of abscisic Acid, cytokinin, and auxin content in salt-stressed plant tissues.

Plant hormones cytokinins, auxin (indole-3-acetic acid), and abscisic acid are central to regulation of plant growth and defence to abiotic stresses such as salinity. Quantification of the hormone levels and determination of their ratios can reveal different plant strategies to cope with the stress, e.g., suppression of growth or mobilization of plant metabolism. This chapter describes a procedure enabling such quantification. Due to the high variability of these hormones in plant tissues, it is advantageous to determine their content in the same sample. Reverse phase and ion exchange chromatography allows separation of the individual hormone fractions. Hormones as well as their metabolites can be identified and quantified by LC/MS.

Indole-3-acetic acid biosensor based on G-rich DNA labeled AuNPs as chemiluminescence probe coupling the DNA signal amplification.

A highly sensitive chemiluminescence (CL) method for detection of phytohormone indole-3-acetic acid (IAA) was developed by using G-rich DNA labeled gold nanoparticles (AuNPs) as CL probe coupling the DNA signal amplification technology. The IAA antibody was immobilized on carboxyl terminated magnetic beads (MBs). In the presence of IAA, antibody labeled AuNPs were captured by antibody functionalized MBs. The DNA on AuNPs is released by a ligand exchange process induced by the addition of DTT. The released DNA is then acted as the linker and hybridized with the capture DNA on MBs and probe DNA on AuNPs CL probe. The CL signal is obtained via the instantaneous derivatization reaction between a specific CL reagent, 3,4,5-trimethoxyl-phenylglyoxal (TMPG), and the G-rich DNA on AuNPs CL probe. IAA can be detected in the concentration range from 0.02 ng/mL to 30 ng/mL, and the limit of detection is 0.01 ng/mL.

Sucrose accelerates flower opening and delays senescence through a hormonal effect in cut lily flowers.

Sugars are generally used to extend the vase life of cut flowers. Such beneficial effects have been associated with an improvement of water relations and an increase in available energy for respiration by floral tissues. In this study we aimed at evaluating to what extent (i) endogenous levels of sugars in outer and inner tepals, androecium and gynoecium are altered during opening and senescence of lily flowers; (ii) sugar levels increase in various floral tissues after sucrose addition to the vase solution; and (iii) sucrose addition alters the hormonal balance of floral tissues. Results showed that endogenous glucose levels increased during flower opening and decreased during senescence in all floral organs, while sucrose levels increased in outer and inner tepals and the androecium during senescence. Sucrose treatment accelerated flower opening, and delayed senescence, but did not affect tepal abscission. Such effects appeared to be exerted through a specific increase in the endogenous levels of sucrose in the gynoecium and of glucose in all floral tissues. The hormonal balance was altered in the gynoecium as well as in other floral tissues. Aside from cytokinin and auxin increases in the gynoecium; cytokinins, gibberellins, abscisic acid and salicylic acid levels increased in the androecium, while abscisic acid decreased in outer tepals. It is concluded that sucrose addition to the vase solution exerts an effect on flower opening and senescence by, among other factors, altering the hormonal balance of several floral tissues.

Simultaneous determination of different endogenetic plant growth regulators in common green seaweeds using dispersive liquid-liquid microextraction method.

A simple and rapid HPLC-based method was developed for simultaneous determination of major classes of plant growth regulators (PGRs) in Monostroma and different species of Ulva. The plant growth regulators determined included gibberellic acid (GA(3)), indole-3-acetic acid (IAA), abscisic acid (ABA), indole-3-butyric acid (IBA), salicylic acid and kinetin riboside (KR) and their respective elution time was 2.75, 3.3, 3.91, 4.95, 5.39 and 6.59 min. The parameters optimized for distinct separation of PGRs were mobile phase (60:40 methanol and 0.6% acetic acid in water), column temperature (35°C) and flow rate (1ml/min). This method presented an excellent linearity (0.2-100µg/ml) with limit of detection (LOD) as 0.2µg/ml for ABA, 0.5µg/ml for KR and salicylic acid, and 1µg/ml for IAA, IBA and GA(3). The precision and accuracy of the method was evaluated after inter and intra day analysis in triplicates. The effect of plant matrix was compensated after spiking and the resultant recoveries estimated were in the range of 80-120%. Each PGR thereby detected were further characterized by ESI-MS analysis. The method optimized in this study determined IBA along with IAA for the first time in the seaweed species investigated except Ulva linza where the former was not detected. In all the species studied, ABA level was detected to be the highest while kinetin riboside was the lowest. In comparison to earlier methods of PGR analysis, sample preparation and analysis time were substantially reduced while allowing determination of more classes of PGRs simultaneously.

The metabolic and developmental roles of carotenoid cleavage dioxygenase4 from potato.

The factors that regulate storage organ carotenoid content remain to be fully elucidated, despite the nutritional and economic importance of this class of compound. Recent findings suggest that carotenoid pool size is determined, at least in part, by the activity of carotenoid cleavage dioxygenases. The aim of this study was to investigate whether Carotenoid Cleavage Dioxygenase4 (CCD4) activity affects potato (Solanum tuberosum) tuber carotenoid content. Microarray analysis revealed elevated expression of the potato CCD4 gene in mature tubers from white-fleshed cultivars compared with higher carotenoid yellow-fleshed tubers. The expression level of the potato CCD4 gene was down-regulated using an RNA interference (RNAi) approach in stable transgenic lines. Down-regulation in tubers resulted in an increased carotenoid content, 2- to 5-fold higher than in control plants. The increase in carotenoid content was mainly due to elevated violaxanthin content, implying that this carotenoid may act as the in vivo substrate. Although transcript level was also reduced in plant organs other than tubers, such as leaves, stems, and roots , there was no change in carotenoid content in these organs. However, carotenoid levels were elevated in flower petals from RNAi lines. As well as changes in tuber carotenoid content, tubers from RNAi lines exhibited phenotypes such as heat sprouting, formation of chain tubers, and an elongated shape. These results suggest that the product of the CCD4 reaction may be an important factor in tuber heat responses.

Direct analysis of abscisic acid in crude plant extracts by liquid chromatography--electrospray/tandem mass spectrometry.

A new method, based on liquid chromatography--electrospray/tandem mass spectrometry, for the determination of abscisic acid (ABA), an essential plant hormone that regulates key metabolic pathways and responses to environmental cues, has been developed. Substantial changes in extraction procedures are also proposed. Data indicate that the organic solvents classically used as extraction buffers can be substituted by an aqueous solution, resulting in the same amounts of extracted ABA. The new method, which uses minute amounts of plant tissue, has an estimated limit of detection below 50 pmol ABA/g, and the sensitivity of the technique allows the analysis of ABA in crude plant extracts. Overall, this new rapid, sensitive and accurate procedure to determine ABA will allow analysis of multiple samples in a short time and represents a clear advantage in comparison with the conventional procedures involving many preparative steps and large amounts of plant tissue.

Charcoal affects early development and hormonal concentrations of somatic embryos of hybrid larch.

Embryogenic tissue of hybrid larch (Larix x marschlinsii Coaz) was multiplied on Medium M (modified MSG medium supplemented with the plant growth regulators (PGRs) 2,4-dichlorophenoxyacetic acid (2,4-D; 9 microM) and N-6-benzyladenine (2.25 microM)). After 1 week, cultures were transferred to either MSG lacking PGRs (Medium C-) or MSG lacking PGRs but supplemented with 1% activated charcoal (Medium C+). Embryos were sampled after 1 week on Medium M, C- or C+. Embryos were analyzed by ELISA for abscisic acid (ABA), abscisic acid-glucose ester, 2,4-D, indole-3-acetic acid (IAA), indole-3-aspartate (IAAsp), zeatin (Z), zeatin riboside (ZR), isopentenyladenine (iP) and isopentenyladenosine (iPA). Transfer of embryos to Medium C+ reduced the embryo concentrations of 2,4-D and iPA, but resulted in elevated concentrations of IAA, IAAsp, ABA, Z, ZR and iP. Charcoal reduced 2,4-D concentrations of embryos by an order of magnitude greater than PGR-free medium alone. Charcoal affected embryo concentrations of five of the eight PGRs quantified. Use of either C+ or C- medium as part of the maturation protocols also affected germination and plantlet establishment of the embryos. A 1-week treatment on Medium C+ positively influenced plantlet establishment and generally reduced variability during both germination and plantlet establishment.

[Studies on the plant hormones produced by 5 species of endophytic fungi isolated from medicinal plants (Orchidacea)].

OBJECTIVE: To study the plant hormones produced by 5 species of endophytic fungi isolated from medicinal plants and to illustrate the mechanism on endophytic fungi stimulating the growth of plants. METHODS: Extracting plant hormones from mycelia and its culture solution with organic solvent, and detecting them by HPLC. RESULTS: One or more plant hormones [GA3 (Gibberellin), IAA (Indoleacetic acid), ABA (Abscisic acid), Z (Zeatin), ZR (Zeatin riboside)] were detected from the mycelia and its culture solution. CONCLUSIONS: The plant hormones produced by the endophytic fungi are important materials that may be used to reveal the mechanism of endophytic fungi stimulating the growth of medicinal plants (Orchidacea).

Ultrastructural evidence of abscisic acid binding sites in guard cells of Vicia faba

Se presenta el uso de una técnica citoquímica que utiliza anticuerpo contra ácido abscísico (ABA) unido al complejo peroxidasa-antiperoxidasa (PAP) por globulina de oveja anti-conejo, para localizar sitios de unión de ABA a nivel de la membrana plasmática, mediante microscopía electrónica. La identificación de las características moléculas cíclicas de PAP confirma, en alta resolución, la tinción específica en secciones ultrafinas, a lo largo de la membrana plasmática de protoplastos de células estomáticas de Vicia faba. La intensidad de la tinción inmunocitoquímica sobre la membrana plasmática que enfrenta al apoplasto fue mayor en especímenes incubados con ABA 10**4 M, así como en aquellos sometidos a estrés hídrico, en comparación con los controles. Mediante el uso de este método sensible, se presenta evidencia ultraestructural del hipotético sitio de acción del ABA en las células estomáticas

Ultrastructural evidence of abscisic acid binding sites in guard cells of Vicia faba

Se presenta el uso de una técnica citoquímica que utiliza anticuerpo contra ácido abscísico (ABA) unido al complejo peroxidasa-antiperoxidasa (PAP) por globulina de oveja anti-conejo, para localizar sitios de unión de ABA a nivel de la membrana plasmática, mediante microscopía electrónica. La identificación de las características moléculas cíclicas de PAP confirma, en alta resolución, la tinción específica en secciones ultrafinas, a lo largo de la membrana plasmática de protoplastos de células estomáticas de Vicia faba. La intensidad de la tinción inmunocitoquímica sobre la membrana plasmática que enfrenta al apoplasto fue mayor en especímenes incubados con ABA 10**4 M, así como en aquellos sometidos a estrés hídrico, en comparación con los controles. Mediante el uso de este método sensible, se presenta evidencia ultraestructural del hipotético sitio de acción del ABA en las células estomáticas (AU)