Roy O. Morris

Combined high-performance liquid chromatography-radioimmunoassay for cytokinins

Abstract The cytokinins isopentenyladenosine and ribosylzeatin were conjugated to bovine serum albumin and the conjugates used to raise antisera in rabbits. The resulting antisera had high specificity towards the cytokinin haptens and low cross-reactivity towards other purines. They were used as the basis for a radioimmunoassay for cytokinins, which, when applied in conjunction with high-performance liquid chromatography, allowed rapid and sensitive (to the picogram range) estimation and identification of multiple cytokinins from natural plant and bacterial sources.

Tzs, a nopaline Ti plasmid gene from Agrobacterium tumefaciens associated with trans-zeatin biosynthesis

SummaryThe tzs gene, present in nopaline Ti plasmids, confers on Agrobacterium tumefaciens the ability to produce the phytohormone, trans-zeatin (Regier and Morris (1982) Biochem Biophys Res Comm 104:1560–1566). This gene has now been cloned from the nopaline Ti plasmid pTiC58. It occurs outside the T-DNA in a region close to that associated with virulence functions. Sequence studies indicate that tzs has substantial homology with the T-region gene, ipt, which is known to encode a dimethylallylpyrophosphate transferase, the first enzyme of the cytokinin biosynthetic pathway. As expected from its homology with ipt, tzs possesses significant DMA transferase activity but when expressed in Escherichia coli it causes secretion of trans-zeatin.

Analysis of picogram quantities of indole-3-acetic acid by high performance liquid chromatography-fluorescence procedures

The use of spectrofluorimeter coupled to a reverse phase high performance liquid chromatography column permits selective detection of indole-3-acetic acid at the low picogram level. The value of the technique is demonstrated by the analysis of endogenous IAA in elongating shoots, xylem sap and callus of Douglas-fir. The data are also used to illustrate a procedure whereby the accuracy of chromatographic analyses can be verified within definable probability limits.

Trans-zeatin in culture filtrates of Agrobacterium tumefaciens

Abstract Cytokinin-active bases and nucleosides have been isolated from the culture filtrates of Agrobacterium tumefaciens by trace enrichment onto octadecyl-silica and have been identified by GLC-MS of their permethyl and trimethylsilyl derivatives. Besides the expected 6-(3-methylbut-2-enylamino) purine, the filtrate contained zeatin (85% trans, 15% cis), 2-methylthio-ribosylzeatin and smaller quantities of ribosylzeatin and other cytokinin-active nucleosides.

Genes Specifying Auxin and Cytokinin Biosynthesis in Prokaryotes

Since the discovery and characterization of auxins and cytokinins, much time, effort and resources have been expended in order to determine the mechanism of their biosynthesis and mode of action. It would not be an understatement to say that the effort has met with only moderate success. For example, in the case of the cytokinins, the structures are known (46), their metabolism has been extensively documented (29) and the major morphological and physiological consequences of their action have been described (26). Nevertheless, there are large gaps in our understanding. Biosynthesisis a case in point. Although there have been reports of isolation of putative cytokinin biosynthetic enzymes (9,10), no enzyme has been obtained in a state of analytical homogeneity and there is no detailed understanding of the biosynthetic pathway, its expression in different plant organs and its control. The genes which encode the biosynthetic enzymes have yet to be isolated and important questions relating to the temporal and spatial expression of such genes during development remain to be answered.

Hormonal Regulation of Seed Development

Shortly after pollination, developing cereal grains exhibit significant transient increases in two of the major plant hormones, auxins and cytokinins. A peak in active cytokinins occurs between three to eight days after pollination in all species examined so far. Both zeatin and zeatin riboside concentrations increase by as much as one hundred fold for a period of three days and then decrease equally rapidly. The peak always coincides in time with the peak in endosperm cell division rate, although no causal relationship has been demonstrated between the two events. The enzyme cytokinin oxidase increases shortly after the increase in active cytokinins and is responsible for reducing cytokinins back to basal levels. The cytokinin peak is followed by a peak in free indole-3-acetic acid (approximately 10 to 15 days after pollination) and is associated with the endosperm cell enlargement and endoreduplication phase. The high levels of free IAA are not destroyed but are converted to various conjugates which probably supply the developing seedling with a source of free IAA.

Secretion of trans-zeatin by Agrobacterium tumefaciens: A function determined by the nopaline Ti plasmid

Abstract Production of the plant cytokinin, trans -zeatin, by a number of strains of Agrobacterium tumefaciens was measured by a combination of traceenrichment, HPLC and radioimmunoassay and confirmed by mass spectrometry. Secretion of trans -zeatin into a culture medium is a constitutive function of those strains which harbor a nopaline Ti plasmid. Strains cured of the nopaline Ti plasmid and those which harbor octopine or agropine plasmids are non-producers. Reacquisition of nopaline plasmids by cured strains restores production.

Phage-displayed peptides as developmental agonists for Phytophthora capsici zoospores.

ABSTRACT As part of its pathogenic life cycle, Phytophthora capsici disperses to plants through a motile zoospore stage. Molecules on the zoospore surface are involved in reception of environmental signals that direct preinfection behavior. We developed a phage display protocol to identify peptides that bind to the surface molecules of P. capsici zoospores in vitro. The selected phage-displayed peptides contained an abundance of polar amino acids and proline but were otherwise not conserved. About half of the selected phage that were tested concomitantly induced zoospore encystment in the absence of other signaling agents. A display phage was shown to bind to the zoospore but not to the cyst form of P. capsici. Two free peptides corresponding to active phage were similarly able to induce encystment of zoospores, indicating that their ability to serve as signaling ligands did not depend on their exact molecular context. Isolation and subsequent expression of peptides that act on pathogens could allow the identification of receptor molecules on the zoospore surface, in addition to forming the basis for a novel plant disease resistance strategy.

Efficacy of different Agrobacterium tumefaciens strains in transformation of pinaceous gymnosperms

Abstract The ability of a series of wild-type and transconjugant Agrobacterium tumefaciens and Agrobacterium rhizogenes strains to incite galls on four pinaceous gymnosperm hosts was determined. The hosts were Pseudotsuga menziesii, Pinus ponderosa, Tsuga heterophylla , and Abies procera . Transformation was confirmed by identification of gall opines. The frequency with which galls were incited depended upon the identity of the infecting strain and the identity of the host. Several strains were highly virulent on all hosts. With one exception, an agropine strain, these strains harboured nopaline Ti plasmids. Restriction enzyme digestion and gel electrophoresis of a set of nopaline Ti plasmids from among the highly virulent strains showed that they differed significantly from each other. Such strains may serve as a basis for the construction of efficient vectors for gymnosperm transformation.

Trace enrichment of cytokinins from Douglas-fir xylem extrudate

SummaryQuantitative trace-enrichment of cytokinins from a plant source (xylem sap from the Douglasfir, Pseudotsuga menziesii (Mirb.) Franco) has been achieved by adsorption onto octadecyl-silica columns followed by elution with ethanol. Adsorption is rapid and efficient and allows complete recovery of cytokinins at the nanomolar level. Douglas-fir sap contains at least four compounds having cytokinin activity, one of which co-elutes with zeatin and another with ribosylzeatin.