Henri A. Van Onckelen

Micro and capillary liquid chromatography-tandem mass spectrometry : a new dimension in phytohormone research

Abstract Quantification of phytohormones in small amounts of material requires sensitive analytical techniques. The analysis of indoles and cytokinins using electrospray tandem mass spectrometry (MS–MS) was described earlier. Gradient elution resulted in an improved detection limit. Micro liquid chromatography (LC) and capillary LC, in combination with large volume injections, resulted in a 200 to 1200 times net increase in sensitivity in comparison with conventional isocratic LC–MS–MS. We obtained a linearity range between 1 fmol and 5 pmol for capillary LC–MS–MS and between 5 fmol and 1 nmol for micro LC–MS–MS.

Ion-pair liquid chromatography–electrospray mass spectrometry for the analysis of cyclic nucleotides

An LC-MS method has been developed combining ion-pair chromatography with an electrospray interface linking microbore and capillary HPLC to mass spectrometry. Separation of cyclic nucleotides on C18 reversed-phase columns, using tetrabutylammonium bromide as an ion pairing agent was evaluated with different mobile phase compositions. It was found that low ion-pairing agent concentration (50-500 microM) used in combination with low flow-rates (5-10 microl min(-1)) allowed the system to operate for up to several days without observing a reduced signal caused by source pollution. The loss of sensitivity expected in ion-pair chromatography could be remedied by using a 2-propanol coaxial sheath flow. Optimal conditions for negative ion electrospray resulted in a linear detection response in the femtomole to picomole range. Using biological samples this method was evaluated and compared with a classical ion-suppression RP-HPLC method using UV detection.

STIMULATION OF INDOLE-3-ACETIC ACID PRODUCTION IN RHIZOBIUM BY FLAVONOIDS

Flavonoids activate nod gene expression in Rhizobium resulting in the synthesis of Nod signals which trigger organogenesis in the host plant. This paper shows that nod‐inducers also stimulate the production of the phytohormone IAA (indole‐3‐acetic acid).

Identification of cytokinins produced by the plant parasitic nematodes Heterodera schachtii and Meloidogyne incognita

SUMMARY The presence of different types of cytokinins was analysed in exudates and lysates of stage-2 juveniles of Heterodera schachtii and Meloidogyne incognita and in mixed stages of Caenorhabditis elegans. For all species, cytokinins were detected in lysates and exudates in which benzyladenine and zeatin-type cytokinins were the most prominent forms. The production of cytokinins by Meloidogyne was much higher than by Heterodera, and the detected levels were in a range which interfered with the physiological activities of the host plant. The presence of 5-methoxy-N,N-dimethyltryptamine hydrogen oxalate did not affect hormone production by H. schachtii, whereas resorcinol slightly stimulated hormone production by M. incognita. The exuded cytokinins may play a role in feeding site induction, more particularly in cell cycle activation and in establishing the feeding site as a nutrient sink.

HPLC Linked Electrospray Tandem Mass Spectrometry: A Rapid and Reliable Method to Analyse Indole‐3‐Acetic Acid Metabolism in Bacteria

Electrospray tandem mass spectrometry in combination with high-performance liquid chromatography can be used for the simultaneous detection and quantification of indole-3-acetic acid, indole-3-methanol, tryptophan, indole-3-acetamide, indole-3-ethanol, tryptamine, indole-3-acetaldehyde, indole-3-acetonitrile, indole-3-aldehyde, indole-3-lactic acid, indole-3-pyruvate and anthranilate on an individual basis. Although methylation is not an absolute prerequisite for analysis of most carboxyl-type indole compounds, samples were methylated before analysis and this improved the detection limits for tryptophan, indole-3-acetic acid, anthranilate and indole-3-lactate up to 1000-fold. Moreover, owing to this methylation no ion suppression conditions, necessary for chromatography of organic acids, have to be used and stability of indole-3-pyruvate was achieved. A detection limit of 100 fmol indole-3-acetic acid methyl ester injected on-column was obtained under multiple reaction monitoring conditions. In view of the analysis of a large number of samples, the chromatographic conditions selected are a compromise between speed of analysis and resolution.

Quantitation of Cyclic Nucleotides in Biological Samples by Negative Electrospray Tandem Mass Spectrometry Coupled to Ion Suppression Liquid Chromatography

Measurement of cyclic nucleotides by electrospray ionization mass spectrometry constitutes an unequivocal quantitative and qualitative technique superior to the established estimation methods of UV spectroscopy, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) and other mass spectrometric ionization techniques such as electron impact, thermospray and fast-atom bombardment. Optimal conditions for negative-ion electrospray tandem mass spectrometry showed a linear detection range using solutions with a concentration ranging from 10−6 and 5×10−9 M with an absolute detection limit of 100 fmol injected. Measurement of diagnostic product ions derived from low-energy collision-activated dissociation of the [M−H]− ions provided unambiguous identification. Quantitation of cyclic nucleotides present in biological samples in the femtomole region, needed further purification using on-line ion suppression high-performance liquid chromatography in order to avoid reduced sensitivity caused by matrix quenching.

Analysis of cyclic nucleotides and cytokinins in minute plant samples using phase-system switching capillary electrospray-liquid chromatography-tandem mass spectrometry

Using an electrospray tandem mass spectrometer as a concentration-sensitive detector, a method has been developed to quantify femtomole amounts of plant growth regulators (i.e. isoprenoid type cytokinins, zeatin, dihydrozeatin, isopentenyladenine and their respective riboside and glucoside analogues) and the second messenger adenosine 3′:5′-cyclic monophosphate (3′:5′-cAMP). Miniaturisation of the chromatographic setup using capillary high performance liquid chromatographic (HPLC) ion spray mass spectrometry increased the sensitivity to the low femtomole region. Application of automated capillary column switching allowed the introduction of large injection volumes into the HPLC system. Aliquots (25 µL) were injected into one dimension of the HPLC set-up and stacked onto a micro pre-column. By means of mobile phase switching the pre-column was back-flushed to introduce the analytes onto the analytical column. For cytokinin analysis positive electrospray ionisation was used and resulted in 2.5–25 fmol detection limits. Cyclic nucleotides were separated under ion-pair conditions using tetrabutyl ammonium bromide as ion-pair reagent and were detected under negative electrospray ionisation conditions. Here a 25 fmol detection limit was determined. Following this approach, cytokinins and 3′:5′-cAMP extracted from only mg amounts of apical shoot meristem and chloroplasts obtained from Nicotiana tabacum cv. Petit Havana SR1 were identified and quantified. Copyright

The effect of an elevated cytokinin level using the **ipt** gene and N^{6} -benzyladenine on single node and intact potato plant tuberization **in vitro**

Two models of potato (Solanum tuberosum L.) tuberization in vitro (intact plants and single nodes) were used to study the role of cytokinins in this process. We applied hormone in two different ways. The exogenous addition of 10 mg · L-1N6-benzyladenine (BA) into the tuberization medium resulted in advanced tuber formation in intact plants, and microtubers appeared 10–20 days earlier than in the experiments in which no cytokinin was supplied. Transformation with the Agrobacterium tumefaciens ipt gene provided potato clones with endogenously elevated cytokinin levels (3–20 times higher zeatin riboside content in different clones). The onset of tuberization in intact ipt-transformed plants with low transgene expression was advanced in comparison with control material, and exogenously applied BA further promoted the tuberization process. On the contrary, tuberization was strongly inhibited in ipt-transformed nodes, and an external increase of the cytokinin level caused complete inhibition of expiant growth. In untransformed (control) nodes cytokinin application resulted in primary and secondary tuber formation, which depended on the BA concentration in cultivation media.

IAA synthesis and root induction with iaa genes under heat shock promoter control.

We have devised a heat shock-inducible indole-3-acetic acid (IAA) synthesis system for plant cells, which is based on the iaa genes of the Agrobacterium tumefaciens T-DNA and the heat shock promoter hsp70 of Drosophila melanogaster.Two DNA constructs were tested: one contains the iaaM gene linked to the hsp70 promoter (hsp 70-iaaM) and encodes the production of indoleacetamide (IAM), the other contains hsp 70-iaaM and the wild-type iaaH gene which codes for the conversion of IAM into IAA (hsp 70-iaaM/iaaH). Heat shock-controlled IAM and IAA synthesis was tested on two levels: biochemically by measuring IAM and IAA levels in Kalanchoe stem segments infected with the two constructs, and morphologically by IAA-dependent root formation on Kalanchoe plants, on carrot discs and on tobacco leaf fragments. At both levels the responses were found to be controlled by the heat shock promoter. IAM levels of segments infected with hsp 70-iaaM increased 6-fold upon heat shock induction to 240 pmol IAM per stem segment. The accumulation of IAA in segments infected with hsp 70-iaaM/iaaH and heat-shocked was found to be more variable, possibly due to IAA transport and metabolism. Heat shock treatment of Kalanchoe plants and tobacco leaf fragments infected with hsp 70-iaaM/iaaH led to a strong increase in root formation. On carrot discs, heat shock-specific root induction was also demonstrated, but the responses differed between individual carrots.

Extraction and quantitative analysis of 1-aminocyclopropane-1-carboxylic acid in plant tissue by gas chromatography coupled to mass spectrometry

We developed a new method for the determination of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) using quantitative GC-negative chemical ionisation MS as a detection and quantification system, in combination with isotope dilution using [2H4]ACC and an off-line solid-phase extraction. By derivatisation with pentafluorobenzyl bromide, ACC could easily be detected with m/z 280 being the most abundant ion. Determination of this component resulted in a detection limit of 10 fmol and a linear fit in the 100 fmol-100 pmol range. The combination of a rapid, high yield purification method with a stable derivatisation procedure and a sensitive detection method allowed the detection of ACC in samples as low as 100 mg fresh mass.