Roger Horgan

A novel strategy for production of a highly expressed recombinant protein in an active form

Under standard growth conditions, E. coli transformed with the high‐level expression vector pMON5525 produces recombinant DMAPP/AMP transferase in inactive, insoluble complexes. We have produced large amounts of active, soluble protein by growing and inducing the cells under osmotic stress in the presence of sorbitol and glycyl betaine. This caused an increase of up to 427‐fold in the active yield, and the disappearance of the protein from the pelletable fraction of cell extracts. This treatment may have wide applicability.

The carotenoid and abscisic acid content of viviparous kernels and seedlings ofZea mays L.

Carotenoid and abscisic acid (ABA) levels were determined in endosperm, embryos and seedlings of wild-type and viviparous (vp) mutants ofZea mays L. Carotenoid concentrations were determined by absorption spectrometry following purification by high-performance liquid chromatography and ABA concentrations by combined gas chromatography-mass spectrometry. Lutein and zeaxanthin were the terminal carotenoids in wild-type tissue. The carotenoid profiles ofvp-1 andvp-8 tissue were similar to that of the wild type; invp-2, vp-5, vp-7 andvp-9 carotenogenesis was blocked at early stages so that xanthophylls were absent. Except forvp-1, where the ABA content was similar to the wild type, the ABA content ofvp embryos was substantially reduced, to 6–16% of the corresponding wild type. Thus, the absence of xanthophylls was associated with reduced ABA content, which was in turn correlated with vivipary. Kernels ofvp-8 had a reduced ABA content although xanthophylls were present. Seedlings of carotenoid-deficient mutants rescued from viviparous kernels contained less ABA than did wild-type seedlings grown in the same way. Furthermore, the ABA concentration of such seedlings did not increase in response to water deficit. Conversely,vp-1 seedlings contained normal levels of carotenoids and ABA. Carotenoid-deficient seedlings did not contain appreciable amounts of chlorophyll so that chloroplast development was not normal. Thus ABA-deficiency could be associated with abnormal plastid development rather than the absence of carotenoids per se.

The effect of auxin concentration on cytokinin stability and metabolism

The stability of [3H]zeatin riboside supplied to freshly excised tobacco pith explants was found to be inversely related to α-naphthaleneacetic acid concentration in the incubation medium. At higher concentrations of α-naphthaleneacetic acid greater breakdown of [3H]zeatin riboside was indicated by higher levels of degradative metabolites (adenine, adenosine and adenosine nucleotides) formed. This auxin effect on cytokinin metabolism appears to be mediated, at least in part, through cytokinin oxidase. The results of in-vitro assays carried out with partially purified enzyme from corn kernels substantiale this conclusion. These findings are discussed in relation to recent observations of auxin and cytokinin levels in crown-gall tumours with altered morphology.

High-performance liquid chromatography of cytokinins

Abstract Details are given of reversed-phase, normal-phase, and adsorption high-performance liquid chromatographic systems suitable for the separation of a wide range of naturally occurring cytokinins related to zeatin. Particular attention has been given to the development of systems to separate cytokinins which are difficult to separate by more conventional means.

The Biosynthesis of Cytokinins in Crown-Gall Tissue of Vinca rosea

The crown-gall tissue of Vinca rosea converts labelled adenine into cytokinins. The principal initial products appear to be ribosylzeatin phosphates; zeatin and ribosylzeatin are also produced in appreciable quantities. The efficiency of conversion of adenine into cytokinins suggests a pathway of synthesis independent of turnover of tRNA. Isopentenyl adenine or its derivatives do not appear to be intermediates in the conversion of adenine to zeatin compounds. Cytokinins in V. rosea turnover rapidly and further metabolism of zeatin derivatives seems to result in their conversion into glucosides which are the main cytokinin active compounds in the tissue.

Cytokinin metabolism in Phaseolus vulgaris L.

The major cytokinins in stems of decapitated, disbudded bean plants have been identified by enzymic degradation, Sephadex LH20 and reversed phase high performance liquid chromatography, and by combined gas chromatography-mass spectrometry as 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9-β-D-ribofuranosylpurine (zeatin riboside), 6-(4-hydroxy-3-methylbutylamino)-9-β-D-ribofuranosylpurine (dihydrozeatin riboside), and the 5′-phosphates of these compounds (zeatin ribotide and dihydrozeatin ribotide). Minor cytokinins in this tissue were tentatively identified as dihydrozeatin-O-β-D-glucoside and zeatin ribotide-O-β-D-glucoside. [8-14C-]Dihydrozeatin appeared to be rapidly metabolized to dihydrozeatin ribotide when supplied to segments of stems from decapitated plants. These results are discussed in relation to the metabolism and distribution of cytokinins in the whole plant.

A cytokinin glucoside from the leaves of Phaseolus vulgaris L.

Phaseolus vulgaris plants decapitated above the primary leaves accumulate high cytokinin activity. The major cytokinin in these leaves was identified by Sephadex LH 20 chromatography, sensitivity to β-glucosidase and permanganate oxidation, and by combined gas chromatography-mass spectrometry as 6-(4-O-β-D-glucosyl-3-methylbutylamino) purine, (dihydrozeatin-O-β-D-glucoside). A possible reason for the persistance of this compound in the primary leaves is discussed.

The role of cis-carotenoids in abscisic acid biosynthesis.

Evidence has been obtained which is consistent with 9′-cis-neoxanthin being a major precursor of abscisic acid (ABA) in higher plants. A mild, rapid procedure was developed for the extraction and analysis of carotenoids from a range of tissues. Once purified the carotenoids were identified from their light-absorbance properties, reactions with dilute acid, high-performance liquid chromatography Rts, mass spectra and the quasiequilibria resulting from iodine-catalysed or chlorophyllsensitised photoisomerisation. Two possible ABA precursors, 9′-cis-neoxanthin and 9-cis-violaxanthin, were identified in extracts of light-grown and etiolated leaves (of Lycopersicon esculentum, Phaseolus vulgaris, Vicia faba, Pisum sativum, Cicer arietinum, Zea mays, Nicotiana plumbaginifolia, Plantago lanceolata and Digitalis purpurea), and roots of light-grown and etiolated plants (Lycopersicon, Phaseolus and Zea). The 9,9′-di-cisisomer of violaxanthin was synthesised but its presence was not detected in any extracts. Levels of 9′-cis-neoxanthin and all-trans-violaxanthin were between 20- to 100-fold greater than those of ABA in light-grown leaves. The levels of 9-cis-violaxanthin were similar to those of ABA but unaffected by water stress. Etiolated Phaseolus leaves contained reduced amounts of carotenoids (15–20% compared with light-grown leaves) but retained the ability to synthesise large amounts of ABA. The amounts of ABA synthesised, measured as increases in ABA and its metabolites phaseic acid and dihydrophaseic acid, were closely matched by decreases in the levels of 9′-cis-neoxanthin and all-trans-violaxanthin. In etiolated seedlings grown on 50% D2O, deuterium incorporation into ABA was similar to that into the xanthophylls. Relative levels of carotenoids in roots and light-grown and etiolated leaves of the ABA-deficient mutants, notabilis, flacca and sitiens were the same as those found in wild-type tomato tissues.

Cytokinin oxidase fromZea mays kernels andVinca rosea crown-gall tissue.

Cytokinin oxidase has been partially purified from matureZea mays kernels and fromVinca rosea corwn-gall tissue. The molecular weights of the two enzymes, determined by gel filtration, are very different: 94,400 (±10%) forZ. mays and 25,100 (±10%) forV. rosea. Specificity studies have been performed using a large number of synthetic and naturally occurring cytokinins. Only a small number of these compounds serve as substrates and both enzymes exhibit similar substrate specificity. In agreement with other workers, a Δ2 double bond in the N6 side chain is essential for activity. The presence of glucosyl or ribosyl groups in the 7-or 9-position or an alanyl group in the 9-position of the purine moiety have little effect on their susceptibility to cytokinin oxidase, but O-glucosyl derivatives are resistant to oxidation. The relevance of these enzyme systems to studies on cytokinin metabolism and to the endogenous cytokinins is discussed.

Abscisic acid biosynthesis in roots. I: the identification of potential abscisic acid precursors, and other carotenoids

The pathway of water-stress-induced abscisic acid (ABA) biosynthesis in etiolated and light-grown leaves has been elucidated (see A.D. Parry and R. Horgan, 1991, Physiol. Plant. 82, 320–326). Roots also have the ability to synthesise ABA in response to stress and it was therefore of interest to examine root extracts for the presence of carotenoids, including those known to be ABA precursors in leaves. All-trans- and 9′-cis-neoxanthin, all-trans- and 9-cis-violaxanthin, antheraxanthin (all potential ABA precursors), lutein and β-carotene were identified on the basis of absorbance spectra, reactions with dilute acid, retention times upon high-performance liquid chromatography and by comparison with leaf carotenoids that had been analysed by mass spectrometry. The source of the extracted carotenoids was proved to be root tissue, and not contaminating compost or leaf material. The levels of total carotenoids in roots varied between 0.03–0.07% of the levels in light-grown leaves (Arabidopsis thaliana (L.) Heynh, Nicotiana plumbaginifolia Viv., Phaseolus vulgaris L. and Pisum sativum L.) up to 0.27% (Lycopersicon esculentum Mill.). The relative carotenoid composition was very different from that found in leaves, and varied much more between species. All-trans-neoxanthin and violaxanthin were the major carotenoids present (64–91 % of the total), but while Lycopersicon contained 67–80% all trans-neoxanthin, Phaseolus, Pisum and Zea mays L. contained 61–79% all-trans-violaxanthin. Carotenoid metabolism also varied between species, with most of the carotenoids in older roots of Phaseolus being esterified. Roots and leaves of the ABA-deficient aba mutant of Arabidopsis had reduced epoxy-xanthophyll levels compared to the wild-type.