A new approach for cytokinin isolation from Arabidopsis tissues using miniaturized purification: pipette tip solid-phase extraction.
ABSTRACT: UNLABELLED: BACKGROUND:We have developed a new analytical approach for isolation and quantification of cytokinins (CK) in minute amounts of fresh plant material, which combines a simple one-step purification with ultra-high performance liquid chromatography-fast scanning tandem mass spectrometry. RESULTS:Plant tissue samples (1-5?mg FW) were purified by stop-and-go-microextraction (StageTip purification), which previously has only been applied for clean-up and pre-concentration of peptides. We found that a combination of two reverse phases and one cation-exchange phase, was the best tool, giving a total extraction recovery higher than 80%. The process was completed by a single chromatographic analysis of a wide range of naturally occurring cytokinins (bases, ribosides, O- and N-glucosides, and nucleotides) in 24.5 minutes using an analytical column packed with sub-2-microne particles. In multiple reaction monitoring mode, the detection limits ranged from 0.05 to 5 fmol and the linear ranges for most cytokinins were at least five orders of magnitude. The StageTip purification was validated and optimized using samples of Arabidopsis thaliana seedlings, roots and shoots where eighteen cytokinins were successfully determined. CONCLUSIONS:The combination of microextraction with one-step high-throughput purification provides fast, effective and cheap sample preparation prior to qualitative and quantitative measurements. Our procedure can be used after modification also for other phytohormones, depending on selectivity, affinity and capacity of the selected sorbents.
Project description:UNLABELLED: BACKGROUND: Cytokinins (CKs) are a group of plant growth regulators that are involved in several plant developmental processes. Despite the breadth of knowledge surrounding CKs and their diverse functions, much remains to be discovered about the full potential of CKs, including their relationship with the purine salvage pathway, and other phytohormones. The most widely used approach to query unknown facets of CK biology utilized functional genomics coupled with CK metabolite assays and screening of CK associated phenotypes. There are numerous different types of assays for determining CK quantity, however, none of these methods screen for the compendium of metabolites that are necessary for elucidating all roles, including purine salvage pathway enzymes in CK metabolism, and CK cross-talk with other phytohormones. Furthermore, all published analytical methods have drawbacks ranging from the required use of radiolabelled compounds, or hazardous derivatization reagents, poor sensitivity, lack of resolution between CK isomers and lengthy run times. RESULTS: In this paper, a method is described for the concurrent extraction, purification and analysis of several CKs (freebases, ribosides, glucosides, nucleotides), purines (adenosine monophosphate, inosine, adenosine, and adenine), indole-3-acetic acid, and abscisic acid from hundred-milligram (mg) quantities of Arabidopsis thaliana leaf tissue. This method utilizes conventional Bieleski solvents extraction, solid phase purification, and is unique because of its diverse range of detectable analytes, and implementation of a conventional HPLC system with a fused core column that enables good sensitivity without the requirement of a UHPLC system. Using this method we were able to resolve CKs about twice as fast as our previous method. Similarly, analysis of adenosine, indole-3-acetic acid, and abscisic acid, was comparatively rapid. A further enhancement of the method was the utilization of a QTRAP 5500 mass analyzer, which improved upon several aspects of our previous analytical method carried out on a Quattro mass analyzer. Notable improvements included much superior sensitivity, and number of analytes detectable within a single run. Limits of detection ranged from 2 pM for (9G)Z to almost 750 pM for indole-3-acetic acid. CONCLUSIONS: This method is well suited for functional genomics platforms tailored to understanding CK metabolism, CK interrelationships with purine recycling and associated hormonal cross-talk.
Project description:We have developed a N6-dimethylallyladenine (cytokinin) dehydrogenase-based microbiosensor for real-time determination of the family of hormones known as cytokinins. Cytokinin dehydrogenase from Zea mays (ZmCKX1) was immobilised concurrently with electrodeposition of a silica gel film on the surface of a Pt microelectrode, which was further functionalized by free electron mediator 2,6-dichlorophenolindophenol (DCPIP) in supporting electrolyte to give a bioactive film capable of selective oxidative cleavage of the N6- side chain of cytokinins. The rapid electron shuffling between freely diffusible DCPIP and the FAD redox group in ZmCKX1 endowed the microbiosensor with a fast response time of less than 10 s. The immobilised ZmCKX1 retained a high affinity for its preferred substrate N6-(?2-isopentenyl) adenine (iP), and gave the miniaturized biosensor a large linear dynamic range from 10 nM to 10 µM, a detection limit of 3.9 nM and a high sensitivity to iP of 603.3 µAmM-1cm-2 (n?=?4, R2?=?0.9999). Excellent selectivity was displayed for several other aliphatic cytokinins and their ribosides, including N6-(?2-isopentenyl) adenine, N6-(?2-isopentenyl) adenosine, cis-zeatin, trans-zeatin and trans-zeatin riboside. Aromatic cytokinins and metabolites such as cytokinin glucosides were generally poor substrates. The microbiosensors exhibited excellent stability in terms of pH and long-term storage and have been used successfully to determine low nanomolar cytokinin concentrations in tomato xylem sap exudates.
Project description:Cytokinins are plant hormones with biological functions ranging from coordination of plant growth and development to the regulation of senescence. A series of 2-chloro-N6-(halogenobenzylamino)purine ribosides was prepared and tested for cytokinin activity in selected bioassays. Several compounds showed significant activity, especially in delaying senescence in detached wheat leaves. We used microarrays to gather information about the reprogramming of gene transcription when senescent Arabidopsis leaves were treated with selected C2-substituted aromatic cytokinin ribosides that showed high activity in the senescence bioassay. Overall design: Arabidopsis senescent leaves were treated with cytokinins and subsequently used for RNA extraction and hybridization on Affymetrix microarrays. 21-days old Arabidopsis leaves were treated with the appropriate cytokinin or left untreated (DMSO only).
Project description:Cytokinins are plant hormones with biological functions ranging from coordination of plant growth and development to the regulation of senescence. A series of 2-chloro-N6-(halogenobenzylamino)purine ribosides was prepared and tested for cytokinin activity in selected bioassays. Several compounds showed significant activity, especially in delaying senescence in detached wheat leaves. We used microarrays to gather information about the reprogramming of gene transcription when senescent Arabidopsis leaves were treated with selected C2-substituted aromatic cytokinin ribosides that showed high activity in the senescence bioassay. Arabidopsis senescent leaves were treated with cytokinins and subsequently used for RNA extraction and hybridization on Affymetrix microarrays. 21-days old Arabidopsis leaves were treated with the appropriate cytokinin or left untreated (DMSO only).
Project description:The mixed-ligand strategy was selected as an approach to tailor a metal-organic framework (MOF) with microextraction purposes. The strategy led to the synthesis of up to twelve UiO-66-based MOFs with different amounts of functionalized terephthalate ligands (H-bdc), including nitro (-NO2) and amino (-NH2) groups (NO2-bdc and NH2-bdc, respectively). Increases of 25% in ligands were used in each case, and different pore environments were thus obtained in the resulting crystals. Characterization of MOFs includes powder X-ray diffraction, infrared spectroscopy, and elemental analysis. The obtained MOFs with different degrees and natures of functionalization were tested as sorbents in a dispersive miniaturized solid-phase extraction (D-µSPE) method in combination with high-performance liquid chromatography (HPLC) and diode array detection (DAD), to evaluate the influence of mixed functionalization of the MOF on the analytical performance of the entire microextraction method. Eight organic pollutants of different natures were studied, using a concentration level of 5 µg· L-1 to mimic contaminated waters. Target pollutants included carbamazepine, 4-cumylphenol, benzophenone-3, 4-tert-octylphenol, 4-octylphenol, chrysene, indeno(1,2,3-cd)pyrene, and triclosan, as representatives of drugs, phenols, polycyclic aromatic hydrocarbons, and disinfectants. Structurally, they differ in size and some of them present polar groups able to form H-bond interactions, either as donors (-NH2) or acceptors (-NO2), permitting us to evaluate possible interactions between MOF pore functionalities and analytes' groups. As a result, extraction efficiencies can reach values of up to 60%, despite employing a microextraction approach, with four main trends of behavior being observed, depending on the analyte and the MOF.
Project description:Recent and unexpected food alerts about relatively high amounts of pyrrolizidine alkaloids in oregano samples have stressed the need to develop analytical strategies to ensure food safety in this type of foodstuff. Accordingly, this work presents the development of a miniaturized strategy based on the QuEChERS (quick, easy, cheap, effective, rugged and safe) method combined with ultrahigh liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) for the determination of 21 pyrrolizidine alkaloids suggested by the European Food Safety Authority to be monitored in food. The analytical method was properly validated, with overall average recoveries from 77 to 96% and relative standard deviations <13% (n = 9). The method proved to be a sustainable analytical strategy which meets green analytical chemistry principles as it showed good performance by using small amounts of sample (0.2 g), organic solvents (1000 µL), clean-up sorbents (175 mg) and partitioning salts (0.65 g). Its feasibility was verified through the analysis of 23 oregano samples. Of the samples analyzed, 100% were contaminated, with an average concentration of 1254 µg/kg. Lasiocarpine, lasiocarpine N-oxide, europine, europine N-oxide, senecivernine, senecionine, echimidine N-oxide, lycopsamine N-oxide and intermedine N-oxide were the alkaloids which significantly contributed to the contamination of the samples.
Project description:Aminomethylation reactions between chiral amino compounds (S)-(-)-1-phenylethylamine and l-proline with tetranonylresorcinarene and tetra-(4-hydroxyphenyl)resorcinarene in presence of formaldehyde were studied. The reaction between l-proline and resorcinarenes generated regioselectively chiral tetra-Mannich bases, due to the molecular incorporation of the fragment of the chiral amino acid. On the other hand, tetranonylresorcinarene and (S)-(-)-1-phenylethylamine formed regio- and diasteroselectively chiral tetrabenzoxazines, both by chiral auxiliary functionalization and by the transformation of the molecular structure that confers inherent chirality. The products obtained were characterized using IR, 1H-NMR, 13C-NMR, COSY, HMQC, and HMBC techniques. The reaction of (S)-(-)-1-phenylethylamine with tetra-(4-hydroxyphenyl)resorcinarene did not proceed under the experimental conditions. Once the chiral aminomethylated tetra-(4-hydroxyphenyl)resorcinarene was obtained, the chemical modification of poly(GMA-co-EDMA) was studied, and the results showed an efficient incorporation of the aminomethylated compound. For the physical modification, chiral aminomethylated tetranonylresorcinarenes were employed, finding that the incorporation of modified resorcinarenes occurs, but with less efficiency than that observed using chemical modification. The modified polymers were characterized via FT-IR, scanning electron microscopy imaging, and elemental analysis. Finally, polymers modified with chiral resorcinarenes were used as sorbents in norepinephrine microextraction; for practical purposes, artificial urine was prepared and used. To perform the microextraction, the decision was made to use the modern rotating-disk sorptive extraction technique (RDSE), because of its analytical attributes as a green, or eco-friendly, technique. According to the results, the method preliminarily validated for the determination of norepinephrine in artificial urine shows that the modified polymer with chiral derivative of tetra-(4-hydroxyphenyl)resorcinarene worked effectively as a new sorbent phase for the quantitative microextraction of norepinephrine, exhibiting high stability and homogeneity of composition and structure within the working range.
Project description:Cytokinins, which are central regulators of cell division and differentiation in plants, are adenine derivatives carrying an isopentenyl side chain that may be hydroxylated. Plants have two classes of isopentenyltransferases (IPTs) acting on the adenine moiety: ATP/ADP isopentenyltransferases (in Arabidopsis thaliana, AtIPT1, 3, 4-8) and tRNA IPTs (in Arabidopsis, AtIPT2 and 9). ATP/ADP IPTs are likely to be responsible for the bulk of cytokinin synthesis, whereas it is thought that cis-zeatin (cZ)-type cytokinins are produced possibly by degradation of cis-hydroxy isopentenyl tRNAs, which are formed by tRNA IPTs. However, these routes are largely hypothetical because of lack of in vivo evidence, because the critical experiment necessary to verify these routes, namely the production and analysis of mutants lacking AtIPTs, has not yet been described. We isolated null mutants for all members of the ATP/ADP IPT and tRNA IPT gene families in Arabidopsis. Notably, our work demonstrates that the atipt1 3 5 7 quadruple mutant possesses severely decreased levels of isopentenyladenine and trans-zeatin (tZ), and their corresponding ribosides, ribotides, and glucosides, and is retarded in its growth. In contrast, these mutants possessed increased levels of cZ-type cytokinins. The atipt2 9 double mutant, on the other hand, lacked isopentenyl- and cis-hydroxy isopentenyl-tRNA, and cZ-type cytokinins. These results indicate that whereas ATP/ADP IPTs are responsible for the bulk of isopentenyladenine- and tZ-type cytokinin synthesis, tRNA IPTs are required for cZ-type cytokinin production. This work clarifies the long-standing questions of the biosynthetic routes for isopentenyladenine-, tZ-, and cZ-type cytokinin production.
Project description:Crosslinked pyrrolidinium-based poly(ionic liquids) (Pyrr-PILs) were synthesized through a fast, simple, and solventless photopolymerization scheme, and tested as solid phase microextraction (SPME) sorbents. A series of Pyrr-PILs bearing three different alkyl side chain lengths with two, eight, and fourteen carbons was prepared, characterized, and homogeneously coated on a steel wire by using a very simple procedure. The resulting coatings showed a high thermal stability, with decomposition temperatures above 350 °C, excellent film stability, and lifetime of over 100 injections. The performance of these PIL-based SPME fibers was evaluated using a mixture of eleven organic compounds with different molar volumes and chemical functionalities (alcohols, ketones, and monoterpenes). The Pyrr-PIL fibers were obtained as dense film coatings, with 67 ?m thickness, with an overall sorption increase of 90% and 55% as compared to commercial fibers of Polyacrylate (85 ?m) (PA85) and Polydimethylsiloxane (7 ?m) (PDMS7) coatings, respectively. A urine sample doped with the sample mixture was used to study the matrix effect and establish relative recoveries, which ranged from 60.2% to 104.1%.
Project description:Cytokinin receptors play a key role in cytokinin-dependent processes regulating plant growth, development, and adaptation; therefore, the functional properties of these receptors are of great importance. Previously the properties of cytokinin receptors were investigated in heterologous assay systems using unicellular microorganisms, mainly bacteria, expressing receptor proteins. However, within microorganisms receptors reside in an alien environment that might distort the receptor properties. Therefore, a new assay system has been developed allowing studies of individual receptors within plant membranes (i.e. closer to their natural environment). The main ligand-binding characteristics of receptors from Arabidopsis [AHK2, AHK3, and AHK4] and maize (ZmHK1) were refined in this new system, and the properties of full-length Arabidopsis receptor AHK2 were characterized for the first time. Ligand specificity profiles of receptors towards cytokinin bases were comparable with the profiles retrieved in bacterial assay systems. In contrast, cytokinin-9-ribosides displayed a strongly reduced affinity for receptors in the plant assay system, indicating that ribosides as the common transport form of cytokinins have no or very weak cytokinin activity. This underpins the central role of free bases as the sole biologically active cytokinin compounds. According to molecular modelling and docking studies, N (9)-ribosylation alters the bonding pattern in cytokinin-receptor interaction and prevents ?6-?7 loop movement important for tight hormone binding. A common feature of all receptors was a greatly reduced ligand binding at low (5.0-5.5) pH. The particularly high sensitivity of ZmHK1 to pH changes leads to the suggestion that some cytokinin receptors may play an additional role as pH sensors in the lumen of the endoplasmic reticulum.