Raymond S. Burden

Inhibitors of sterol biosynthesis and growth in plants and fungi

Abstract Sterols and their derivatives promote and maintain growth and development in plants and fungi by acting as membrane constituents and probably also as hormones, engaged in control of metabolism. Inhibitors of sterol biosynthesis, operating at various stages in the pathway, are useful probes for investigating these functions. Some of the inhibitors have assumed considerable commercial importance as agricultural fungicides and antimycotic drugs in medicine. There is also potential for using similar compounds to regulate plant growth. The work reviewed here, much of it done within the last few years, points unmistakably to a fundamental role for sterols in promoting growth and development in both plants and fungi. A clear target enzyme in the biosynthetic pathway for growth inhibition in both plants and fungi is the 14α-methylsterol demethylase. Work with triazoles, particularly with closely related analogues and separated enantiomers, has provided an insight into the affinity of these inhibitors for the different forms of cytochrome P-450 which catalyse fungal sterol 14α-demethylation, plant sterol 14α-demethylation and plant ent -kaurene oxidation. It may become increasingly necessary to evaluate the relative contribution of these processes to optimise performance of fungicides, herbicides and PGRs, both with regard to their activity at the primary site of action and with a view to controlling side effects. There is clear scope here for extending and improving in vitro enzyme assay methods. The effectiveness of the morpholines as agricultural fungicides contrasts with the high tolerance of plants to cyclopropyl and Δ 8 -sterols which are readily induced by application of these compounds. Further study of the basic reason for morpholine toxicity is, therefore, an area well worthy of further investigation. Inhibition of squalene epoxidase by allylamines has proved successful in the development of medical antimycotics but no agricultural fungicides, based on this mode of action, have been forthcoming. Other enzymes of the pathway have yet to be commercially exploited but the C-24 alkylation process seems clearly linked to growth in both plants and fungi and hence would appear to be a particularly promising site for new inhibitors. The pre-squalene enzyme HMG-CoA reductase seems to offer potential for regulating or stopping plant growth. Above all there is a need to evaluate precisely how sterols and their derivatives control growth and developmental processes. The judicious use of inhibitors of the type described here may help in this undertaking which, in turn, could generate new ideas for the design and discovery of more active and specific compounds.

Phytoalexins and stress metabolites in the sapwood of trees

Abstract A wide range of organic compounds, many of them fungitoxic or fungistatic, appear in the sapwood of trees after wounding, injury or fungal attack. There is evidence that most of these compounds are formed by dying parenchyma cells and they therefore can be considered to be phytoalexins. In many cases such compounds accumulate in narrow ‘reaction zones’ which serve to impede further progress of pathogens.

Effects of sterol biosynthesis-inhibiting fungicides and plant growth regulators on the sterol composition of Barley plants

Abstract A number of sterol biosynthesis-inhibiting (SBI) fungicides and plant growth regulator analogs were applied as root drenches to barley seedlings and their effect on the total sterol composition of the roots and shoots was measured by gas-liquid chromatography. Prochloraz was found to be inactive in this system, probably because of poor uptake, while the other compounds could be divided into two groups according to their mode of action as assessed by sterol profiling. The morpholines tridemorph and fenpropimorph inhibited the enzyme cycloeucalenol—obtusifoliol isomerase whereas triadimenol, nuarimol, paclobutrazol, and triapenthenol (RSW 0411) inhibited the enzyme responsible for the removal of the C-14 methyl group. Effects of individual diastereo-isomers and enantiomers of some compounds on sterol profiles were compared with their known fungicidal and anti-gibberellin properties. Shoot growth was reduced by all the compounds tested, paclobutrazol, nuarimol, and triapenthenol being the most effective. As well as inducing accumulation of abnormal sterols, SBI fungicide treatment changed the ratio of campesterol to stigmasterol and sitosterol. It is hypothesized that this may reflect changes in membrane architecture and may offer an explanation for the increased frost hardiness sometimes observed with SBI fungicide-treated plants.

Inhibition of celery cell growth and sterol biosynthesis by the enantiomers of paclobutrazol

Abstract The four enantiomeric forms of the triazole plant growth retardant, paclobutrazol, [(2 RS , 3 RS )-1-(4chlorophenyl)-4,4-dimethyl-2-(1H-1,2,4-triazol-1-yl)-pentan-3-ol] were tested as inhibitors of cell growth and sterol composition using a celery suspension culture. The (2 R ,3 R )- and (2 R ,3 S )-enantiomers were potent inhibitors of cell growth and caused a large accumulation of the 14α-methylsterols obtusifoliol, cycloeucalenol, 14α,24-dimethylcholest-8-en-3β-ol and 14α,24-dimethylcholesta-8,24(28)-dien-3β-ol. The (2 S ,3 S )- and (2 S ,3 R )-enantiomers on the other hand were only inhibitory to growth and sterol 14α-demethylation at higher concentrations. It is concluded that the (2 R )-configuration confers the highest potency both for retarding cell proliferation and for inhibition of the cytochrome P-450 dependent sterol 14α-demethylation reaction in celery cells. A lowering of the stigmasterol: sitosterol ratio was noted, particularly with the (2 R ,3 R )- and (2 S ,3 S )-enantiomers, which suggested that the sterol Δ 22 -desaturase may also be a target for inhibition by triazoles. The relevance of these results to the mode of action of (2 RS ,3 RS )-paclobutrazol as a plant growth retardant is discussed.

(−)-7-HYDROXYCALAMENENE, A PHYTOALEXIN FROM TILIA EUROPEA

Abstract The antifungal sesquiterpene (−)-7-hydroxycalamenene has been isolated and identified in Tilia europea (European Lime) infected with the fungus Ganoderma applanatum . It was found in the narrow pigmented boundary zone between healthy and infected wood.

Fluidity and lipid composition of oat and rye shoot plasma membrane: effect of sterol perturbation by xenobiotics

Oat and rye plants were treated with either tetcyclacis (an experimental plant growth regulator), nuarimol (a fungicide) or gamma-ketotriazole (an experimental herbicide). These treatments reduced shoot growth and changed the lipid composition of the shoot plasma membranes. In oat, both tetcyclacis and nuarimol treatments increased plasma membrane cholesterol and increased the phosphatidylethanolamine/phosphatidylcholine (PE/PC) ratio, whereas gamma-ketotriazole treatment reduced cholesterol and the PE/PC ratio. In rye, all treatments reduced the PE/PC ratio. Generally, the sterol/phospholipid ratio was less in oat than in rye but the cholesterol/phospholipid ratio was greater. With all treatments in oat and rye, increases were observed in unsaturation of the phospholipid acyl chains. The fluidity of membranes was measured by steady-state fluorescence polarisation of the probe diphenylhexatriene; oat membranes were more fluid than rye. Membrane fluidity was greater in plasma membranes from plants treated with the xenobiotics than the controls. The results are discussed in the context of the effect of plasma membrane lipid composition on membrane fluidity, and it is concluded that there appears to be no overall simple relationship between membrane lipid composition and fluidity that holds for all treatments in both species.

Sesquiterpene phytoalexins from Ulmus glabra

Abstract Inoculation of Wych elm ( Ulmus glabra ) with the fungi Ceratocystis ulmi , Chrondrostereum purpureum and Coriolus versicolor induced the formation of a series of antifungal cadinane-type sesquiterpenes.

Effects of the growth retardant tetcyclacis on the sterol composition of oat (Avena sativa)

The norbornenodiazetine plant growth regulator tetcyclacis, when applied to roots of Avena sativa, caused a substantial increase in the cholesterol content of the shoots. Amounts of the C-24 alkylated sterols campesterol, stigmasterol and sitosterol all declined. A similar alteration in the sterol profile was observed for a plasma membrane preparation from the shoots. Changes in the sterol composition of root tissue were much less pronounced.

Debneyol, a fungicidal sesquiterpene from tnv infected Nicotiana debneyi

Abstract An antifungal sesquiterpene diol, debneyol, has been isolated from tobacco necrosis virus-inoculated leaves of Nicotiana debneyi and a structure is proposed from chemical, spectroscopic and biogenetic evidence. In contrast to the related phytoalexin capsidiol which is fungistatic, debneyol appears to exhibit genuine fungicidal activity.

Isolation of the ERG2 gene, encoding sterol ?8??7 isomerase, from the rice blast fungus Magnaporthe grisea and its expression in the maize smut pathogen Ustilago maydis

The Magnaporthe grisea ERG2 gene, encoding Δ8Δ→7 sterol isomerase, was isolated from a genomic library by heterologous hybridization to a fragment of the Ustilago maydis ERG2 gene. The isolated gene contained a reading frame of 745 bp which encoded a protein of 221 amino acids. The coding region was interrupted by a single putative 79-bp-long intron. The deduced amino-acid sequence exhibited similarity to the ERG2 gene products of U. maydis and of Saccharomyces cerevisiae, particularly in the central region of the proteins. The NH2-terminal of all three proteins contained a long stretch of amino acids that were strongly hydrophobic, suggesting that they may function by anchoring the protein to a membrane surface. The M. grisea ERG2 gene complemented a U. maydis deletion mutant in which the ERG2 gene had been removed using a one-step gene replacement procedure. The Δ8Δ→7 sterol isomerase produced by the M. grisea ERG2 gene exhibited a level of sensitivity to the sterol biosynthesis inhibitor, tridemorph, similar to that of the enzyme derived from the U. maydis ERG2 gene.