Plant Growth Regulation by Mevinolin and Other Sterol Biosynthesis

Chapter 8

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Plant Growth Regulation by Mevinolin and Other Sterol Biosynthesis Inhibitors Thomas J. Bach and Hartmut K. Lichtenthaler Botanical Institute (Plant Physiology), University of Karlsruhe, Kaiserstrasse 12, D-7500 Karlsruhe, Federal Republic of Germany

There is conclusive evidence that sterols have essential roles in the regulation of growth and development of plants. Mevinolin, a metabolite produced by Aspergillus terreus, specifically inhibits HMG-CoA reductase, a rate limiting enzyme of the isopentenoid pathway. This compound has been used as a molecular probe to study the interrelationships of MVA and sterol biosynthesis in the developmental events of plants and animals. The accumulation of other isopentenoids, such as mitochon drial ubiquinone(s) or plastidic prenylquinones, chlorophylls and carotenoids, is less or not at all affected by mevinolin, thereby emphasizing the concept of mevalonic acid biosynthesis being present in these organelles. The effect on growth of several microbiologically produced or chemically synthesized inhibitors (including mevinolin) known or expected to interfere with enzymes that catalyze steps later in the sterol pathway were evaluated. Generally, the growth inhibitory reactions of treated radish seedlings induced by the various test compounds were quite similar, though some specific reactions induced by mevinolin, such as inhibition of tillering at higher concentrations, suggest a requirement for isopentenoid-derived factors other than sterols to ensure cell division in meristematic plant tissue or in cell cultures. The results indicate that mevinolin may have physiological importance as a plant growth regulator. 0097-6156/87/0325-0109$08.50/0 © 1987 American Chemical Society

Fuller and Nes; Ecology and Metabolism of Plant Lipids ACS Symposium Series; American Chemical Society: Washington, DC, 1987.


110

ECOLOGY AND METABOLISM OF PLANT LIPIDS

A basic question i n which our laboratory has been interested over the l a s t few years revolves around the problem of MVA synthesis and the flow of t h i s compound to the major isopentenoid compounds in the plant c e l l . The pathway f o r the biosynthesis of polyisoprenoids and the p o s i t i o n of the key-regulating enzyme HMG-CoA reductase i s summarized i n Figure 1. Our studies and those of other groups have revealed that the regulatory r o l e of HMG-CoA reductase does not seem to be confined only to mammals (1-8). but can also be extended to plants (9-22) and fungi (23-27). MVA i s the ultimate precursor of s t e r o l s (28) and triterpenoids (29), compounds that act as a r c h i t e c t u r a l and functional components of endoplasmic reticulum and plasmalemma, presumably through s p e c i f i c i n t e r a c t i o n with f a t t y acids and proteins (28, 29). MVA i s also the precursor of prenylquinone components of photosynthetic or oxidative electron transport chains i n the chloroplast and mitochondria (30-33), of isoprenoid l i g h t harvesting accessory pigments such as cartenoids (34), and the mixed prenylpigments chlorophyll a and b (35). That the enzyme HMG-CoA reductase plays an important e c o l o g i c a l r o l e i n growth of plant and microbial c e l l s i s emphasized through the existence of highly s p e c i f i c a n t i b i o t i c s that bind with and regulate t h i s enzyme. A series of m i c r o b i a l l y produced hypocholesterolemic drugs have been i s o l a t e d from d i f f e r e n t s t r a i n s of Ascomycetes (Figure 2). Compactin and the related ML-236A were f i r s t obtained from species of Pénicillium as an antifungal metabolite (36, 37,). Mevinolin has been obtained from A s p e r g i l l u s terreus (38) and Monascus ruber (39). Recently, the isolation of dihydromevinolin from A. terreus (40). dihydrocompactin from P. citrinum (41), monacolins J (42) and X (43) and dihydromonacolin L from M. ruber (43) were reported. These drugs were revealed to be potent competitive i n h i b i t o r s of HMG-CoA reductase and hence of mammalian c h o l e s t r e r o l synthesis, with mevinolin being the most e f f e c t i v e i n h i b i t o r . Compactin (ML-236B) and mevinolin have been found to be extremely u s e f u l tools i n studying the regulation of vertebrate isoprenoid synthesis and i t has been shown that these fungal metabolites s p e c i f i c a l l y i n h i b i t a wide v a r i e t y of prokaryotic and eukaryotic HMG-CoA reductases (37-54) (Table I ) . I t i s i n t e r e s t i n g to note that the biosynthesis of mevinolin and presumably that of related polyketide derived drugs (55-58) seem to begin at a state w i t h i n the fungal l i f e cycle where the accumulation of s t e r o l s and possibly that of other e s s e n t i a l MVAderived products have reached t h e i r stationary phase (52). The possible interference of the mevinolin-type compounds with microorganisms i n the s o i l raises the question as to whether these microorganisms are able to make d e r i v a t i v e s . In systematic studies i t was recently shown that compactin could be β-hydroxylated (59) by Mucor hiemalis at p o s i t i o n 6 (following the numbering system f o r mevinolin as introduced by Alberts et a l . (38) and used i n Figure 2). The nearly exclusive 6 a-hydroxyl a t i o n of compact i n by Synecepha l a s t rum nigricans and S. racemosum (60), however, indicated a high degree of s e l e c t i v i t y and s t e r e o s p e c i f i c i t y of the microbia enzymes (60). In a c e l l - f r e e


BACH AND LICHTENTHALER

11

Plant Growth Regulation

^Acetyl-CoA Flavonoids'


HMG-CoA

I

x

Fatty acids

HO-V^COO® Τ

SCoA MEVINOLIN - < * ~ |«=HMGR CH * Zeatin t-RNA HO-f^COO® / ^ ^ CHo Mevalonate L^OH yWM£H,

ft

3

2

HifJ Η

Figure 2.

THE REDUCED OF B > ,

AND F )

Structures of the f r e e - a c i d forms of mevinolin and of related compounds. The correct absolute configuration i s shown using the nomenclature given by Alberts et a l . (38). Note that one region of the molecules closely resembles the mevaIdyl moiety of (3S,5R)-mevaloyl-CoA thiohemiacetal, the enzyme-bound intermediate in the two-step reduction of (S)-HMG-CoA to (R)-MVA.


Fuller and Nes; Ecology and Metabolism of Plant Lipids ACS Symposium Series; American Chemical Society: Washington, DC, 1987. 1.0 4.5

embryonic Drosophila c e l l s (microsomes) Crithidia fasciculata. semi-purified yeast, p a r t i a l l y

ML-236B

ML-236B

ML-236B

Continued on next page

0.9

homogenates of the Cropora tobacco hornworm (Manduca sexta)

ML-236B

0.24

Monger et a l . 1982 (47)

1.1

human f i b r o b l a s t s , detergent-solubilized

ML-236B

purified

Brown et a l . 1978 (46)

2.66

rat l i v e r microsomes, partially purified

ML-236B

Nakamura & Abeles 1985 (49)

Kim & Holmlund 1985 (50)

Brown et a l . 1983 (48)

Tanzawa & Endo 1979 (45)

Endo et a l . 1976 (44)

10

rat l i v e r microsomes, partially purified

ML-236B ( = Compactin)

Endo et a l . 1976 (44)

Reference

220

Ki (nM)

rat l i v e r microsomes partially purified

Enzyme source

Comparison of i n h i b i t i o n constants (K^ values) of compactin/mevino1intype metabolites against HMG-CoA reductase preparations from various enzyme sources

ML-236A

Compound

Table I.



3.7 ?

rat l i v e r microsomes, partially purified rat l i v e r microsomes, partially purified

4α, 5-Dihydrocompactin

4a»5-Dihydro~ mevinolin

Albers-Schoonberg et a l 1981 (40)

Lam et a l . 1981 (41)

Watson et a l . 1983 (54)

For other compounds ( c f . F i g . 2) no values are a v a i l a b l e as yet. The I50 values reported using r a t l i v e r HMG-CoA reductase (42, 43) appear to be about three magnitudes higher than that of 4ct,5-dihydromevinolin (40).

*50 = 2.7

20

Halobacterium halobium

Mevinolin

Bach & Lichtenthaler 1983 (53)

radish seedlings, microsome-bound

Mevinolin

2.2

yeast, p a r t i a l l y p u r i f i e d

Mevinolin

Bach & Lichtenthaler 1982, 1983 (52, 53)

Endo 1980 (39)

Alberts et a l . 1980 (38)

Reference

3.5

0.50

rat l i v e r microsomes, Mevinolin (= Monacolin Κ) p a r t i a l l y p u r i f i e d

(nM) 0.64

Enzyme source rat l i v e r microsomes, partially purified

Mevinolin

Compound

Table I . Continued


8.


115


system from rat liver the inhibitory activity of 6a-hydroxycompactin carboxylate against cholesterol synthesis was more potent than that of the 6 B-hydroxy-derivative (60). S t r a i n SANK 32772 of Absidia coerulea c a t a l i z e d the conversion of ML-236B to 3a-hydroxy-iso-ML-236B (6a- i n the numbering system used by the Sankyo group (61)). This l a t t e r compound, with ^4,4a | Δ » , was s i m i l a r to the parent compound i n i t s l e v e l of i n h i b i t i o n of cholesterol synthesis (61). Other microbial s t r a i n s capable of 6 β-hydroxylation of ML-236 were ascomycetes i s o l a t e d from s o i l samples c o l l e c t e d i n A u s t r a l i a and were i d e n t i f i e d as substrains of Nocardia autotrophica (62). Several fungal strains (Circinella muscae, Absidia cylindrospora and A. glauca) have been reported to b i o l o g i c a l l y phosphorylate the hydroxy 1 group at p o s i t i o n δ of the open carboxylic forms of compact i n as w e l l as of monacolin Κ (= mevinolin), and monacolins L and X (63). The Basidiomycete Schizophyllum commune was found to transform compact i n as w e l l as mevinolin (monacolin K) to the corresponding 8-a-hydroxy derivatives (64).The existence of a n t i b i o t i c s , other than the mevinolin-type (65), produced by Cephalosporin caerulens (known i n h i b i t o r s of the synthesis of polyketides and of f a t t y acids (66)) indicates a complex biochemical and p h y s i o l o g i c a l interplay may e x i s t among organisms throughout the rhizosphere. This interplay may include higher plants i n the rhizosphere. We have shown that mevinolin, applied as i t s water-soluble sodium s a l t , i n h i b i t s the root growth of i n t a c t radish and wheat seeedlings by i n h i b i t i n g t h e i r isopentenoid biosynthesis (67.). We have u t i l i z e d mevinolin as a molecular probe to study the importance of MVA and associate products to growth and development of seedlings, bearing i n mind that i n vivo i n h i b i t i o n of an enzyme, expected to be at least close to rate l i m i t i n g f o r the pathway, should r e s u l t i n c l e a r morphological and biochemical responses. A logical consequence was to e s t a b l i s h what type of isoprenoid compound might be affected i n i t s synthesis or accumulation i n the presence of mevinolin (52, 68, 69), thereby y i e l d i n g information also on the i n t r a c e l l u l a r l o c a l i z a t i o n of HMG-CoA reductase a c t i v i t y i n plant c e l l s which i s currently a matter of controversy i n l i t e r a t u r e (70-72). Since i t appeared that the accumulation of phytosterols was p r i m a r i l y affected by mevinolin (52, 68, 69) (which led us to hypothesize that mevinolin can e a s i l y penetrate the plant c e l l w a l l and the plasmalemma, but rather poorly the envelopes of mitochondria and plastids), we tested other i n h i b i t o r s known or expected to i n t e r f e r e with l a t e r steps of s t e r o l biosynthesis, such as squalene epoxidation, squalene-oxide c y c l i z a t i o n , 14a-desmethylation or side chain a l k y l a t i o n , i n order to look for similarities and differences i n the morphological response of radish seedlings upon treatment (73). A side aspect of these l a t t e r studies i s to e s t a b l i s h a v e r s a t i l e screening system f o r hypocholesterolemic drugs or fungicides. 5

6


an(

Materials and Methods Chemicals. Mevinolin was k i n d l y provided by Dr. A. W. (Merck Sharp & Dohme Res. Labs.) and was converted

Alberts to i t s


116



water-soluble sodium s a l t as described by K i t a et a l . (74). Triparanol (MER-29), U 18666 A, A 25822 Β were g i f t s from Merrell Dow Pharmaceuticals I n c . , Dr. Harry Rudney (University of Ohio, Cincinnati, OH), and Eli Lilly G.m.b.H., respectively. 2,3-Epiminosqualene was kindly synthesized by Dr. Lunkenheimer (Bayer AG), N a f t i f i n e was purchased from Dr. Hôgenauer (Sandoz Forschungszentrum Wien). Triarimol and imminium s a l t ("NES") were g i f t s of Dr. W. D. Nes (USDA Berkeley). SC 32561 was a g i f t from G. D. Searle & Co. Clotrimazole, miconazole, and sodium deoxycholate were purchased from Sigma. C u l t i v a t i o n of seedlings and associated test systems. Radish seeds (Raphanus s at i vus, cv. Saxa Knacker) were immersed in an aerated water bath for 30 min. These seeds were then placed onto a sprouting tray containing 1 1 of H2O or H2O supplied with the chemicals and were allowed to germinate and develop for one week i n the l i g h t (Osram Fluora lamps 65 W at 2.5 W*m~ , 25°C, 65% r e l a t i v e humidity). Hydrophobic drugs were usually dissolved i n a system containing DMSO:Triton X-100 i n the r a t i o 3:1 ( v : v ) , maximum 1 ml/1 i n the growth solution. Controls contained the same amount of this mixture without i n h i b i t o r s . In some cases, the addition of a small amount of EtOH was required to dissolve the compounds. Plant growth was measured d a i l y (25-30 plants per condition). Wheat seedlings were cultivated from Triticum aestivum cv. Anza. E t i o l a t e d primary leaf segments from one week-old plants were obtained and used i n the test system as previously described (75. Zi>. 2

C e l l Suspension Cultures of Silybum marianum. Suspension cultures were grown from primary c a l l u s cultures. Submersed c e l l s were kept i n Erlenmayer-flasks (120 rpm; 25° C; P h i l l i p s TL 40W/47, 2000 lux) i n a growth medium as described by Murashige and Skoog (77). Four to s i x flasks per condition were inoculated with 5 ml aliquots of a c e l l suspension from 8 to 10 days old c e l l cutures and then supplied with mevinolin to a f i n a l concentration of 0.625, 1.25, 1.5, 5 and 10 μ ϋ . At day 3 after inoculation the c e l l s were in the early log phase of evelopment and the plateau phase was reached between day 5 and 6. Under the culture conditions employed the c e l l s started to degenerate between day 6 and 9. Thus, c e l l s were analyzed at day 3 and 6. Chemical analysis. For the extraction of lipids and prenylpigments the plant material was macerated in the presence of 100% acetone and the l i p i d s partitioned into p e t r o l ether (b.p. 50-70° C). Chlorophylls and t o t a l carotenoid content were determined spectrophotometrically (78). Prenylquinones were separated by TLC, HPLC and reversed phase HPLC as described i n d e t a i l (69). Free 4-desmethylsterols were quantified a f t e r TLC on s i l i c a gel (solvent 84 ml p e t r o l ether b.p. 50-70° C and 15 ml Me2C0, Rf = 0.25). Plates were sprayed with saturated antimony trichloride i n H20-free CHCI3, and a f t e r a short heating period at 100° C, the pink spots indicating desmethylsterols (with pure stigmasterol as a standard) were


8. BACH AND LICHTENTHALER

117


scanned with densimeter (550 nm) being made for each p l a t e .

with separate

standard

curves


Results and Discussion Growth i n h i b i t i o n by mevinolin. To ascertain the s p e c i f i c i t y of mevinolin as an enzyme i n h i b i t o r and to elucidate the r o l e of HMG-CoA reductase a c t i v i t y i n vivo, i t s e f f e c t on plant growth and development i n conjunction with other isopentenoids has been examined. Mevinolin induced a strong growth i n h i b i t i o n of the main root of the dicotyledonous e t i o l a t e d radish seedlings and of the roots of the monocojbyledonous wheat seedlings (67). A s i g n i f i c a n t drop i n elongation growth was obtained between 10 and 100 ppb (2.5 χ 1 0 " and 2.5 χ 10~ M). We suspected that mevinolin inhibited root elongation growth v i a the interference with HMG-CoA reductase a c t i v i t y i n vivo; therefore, the e f f e c t should have been overcome by the uptake of exogenously supplied MVA - the d i r e c t product of the inhibited enzyme reaction. Indeed, increasing concentrations of exogenous MVA i n the presence of mevinolin gave nearly the same growth rate of roots of e t i o l a t e d radish seedlings as found for the control plants (68). Exogenous MVA at an intermediate concentration of 2 mM did not stimulate root elongation growth but rather led to weakly decreased values, probably due to a secondary pH e f f e c t . A main branch point within the isoprenoid pathway i s located at the s i t e of f a m e s y l pyrophosphate (Figure 1). This key p o s i t i o n of C ^ - p r e n y l t r a n s f e r a s e makes i t a l i k e l y candidate for f i n e tuning or rate l i m i t a t i o n of MVA flux into the various end-products, some of which might contribute to normal root (and hypocotyl) growth. Famesol was revealed to enhance root growth i n barley (79, 80). This accelerated root elongation, however, was correlated with increased cytokinin a c t i v i t y as a secondary e f f e c t (79.). This appeared to be to some extent contradictory to the results of Buschmann and Lichtenthaler (81) who demonstrated an inhibitory effect of exogenously supplied kinetin or benzylaminopurine on root elongation of dark- and light-grown radish seedlings. Other related isoprenoid alcohols, however, f a i l e d to enhance the growth of barley roots (80). Furthermore, other plant species tested for t h i s f a r n e s o l - e f f e e t did not exhibit any p o s i t i v e reaction (80). It i s possible that mevinolin might interfere with the synthesis of other isopentenoid phytohormones such as the g i b b e r e l l i n s or the brassinosteroids which are known to induce strong growth promotion ((82-87) and elsewhere i n t h i s volume). We have determined that the growth i n h i b i t i o n of the roots of e t i o l a t e d radish seedlings induced by mevinolin could not be reversed by the addition of g i b b e r e l l i c acid ( G A 3 ) . Exogenously supplied concentrations of GA3 up to 37 uM could not overcome the e f f e c t of mevinolin at 2.5 yM or at 0.25 μΜ (52, 68). Gibberellic acid itself seemed to slightly stimulate root elongation growth of etiolated radish seelings, e s p e c i a l l y i n the l a t e r stages of development [68]. To gain further information about the mode of action of mevinolin, we extended our experiments to light-grown radish 8

7



118


seedlings. In t h i s system, mevinolin proved to be a highly potent i n h i b i t o r of root elongation growth (Figures 3, 4). In e t i o l a t e d seedlings the elongation growth of hypocotyls needs comparably high concentrations of mevinolin to be affected, but in l i g h t - c u l t i v a t e d seedlings i t induces a c l e a r dwarfing growth response. Radish seeds were incubated i n P e t r i dishes on f i l t e r paper with 10 ml solutions of 0, 2.5 μΜ, 0.25 μΜ and .025 μΜ mevinolin (68). Even i n the presence of the i n h i b i t o r at i t s highest concentration, there was no c l e a r e f f e c t on the germination rate (68). The fact that even very high i n h i b i t o r concentrations could not prevent a minimal root growth i n radish seedlings (Figure 3) led us to conclude that t h i s minimal root growth i s a function of MVA-derivatives already present i n the seeds and therefore independent from de novo mevalonate biosynthesis. I t i s evident that increasing concentrations of mevinolin i n h i b i t the elongation growth of main roots i n e t i o l a t e d or light-grown radish seedlings and also block the formation and development of l a t e r a l roots (Figure 4). This might r e f l e c t , as already discussed, an i n t e r a c t i o n of mevinolin with the synthesis of c e r t a i n isopentenoid phytohormones. In addition to the examples c i t e d above, Geuns (88) demonstrated that, besides the stimulation of hypocotyl growth of e t i o l a t e d mung bean seedlings, the l a t e r a l root number could be increased to 50 and/or 250%, respectively, by rather high concentrations (25 to 50 mg.l""*) of corticosterone and C o r t i s o l . C o r t i s o l was found to stimulate root elongation by about 100% and hypocotyl elongation by ' when added to the growth medium at a concentration of 0.41 μ,η (89). Due to the low uptake of C o r t i s o l by the roots, the concentration i n the plant i t s e l f was thought to be i n the range of 0.1 to 1 nM. The growth stimulation was found to be a r e s u l t of c e l l elongation rather than the production of more c e l l s (89). E f f e c t of mevinolin on isoprenoid accumulation i n radish seedlings. Under the conditions i n which mevinolin i s u s u a l l y applied to the seedlings (see Materials and Methods) i t would f i r s t be taken up by the roots exposed to the watery s o l u t i o n of the i n h i b i t o r and then d i s t r i b u t e d to the remaining seedling parts. The e f f e c t of mevinolin on fresh and dry weights, s t e r o l content, and ubiquinone content i n d i f f e r e n t parts of the seedlings upon treatment has been determined (Table I I ) . At a maximum concentration of 5 μΜ, mevinolin reduces the content of free 4-desmethylsterols i n roots to about 20 per cent of the c o n t r o l . At 0.625 μΜ, s t e r o l content i s reduced by about 20 per cent, thus i n d i c a t i n g a very fast response of s t e r o l accumulation to i n h i b i t i o n of MVA biosynthesis. These e f f e c t s are less dramatic i n hypocotyls, and e s p e c i a l l y i n cotyledons. This r e s u l t implies that there might e x i s t a gradient i n the concentration of mevinolin present i n upper parts of the seedlings. The p o s s i b i l i t y of a low transport rate of mevinolin w i t h i n the t i s s u e was indicated when excised dark-grown radish seedlings were used to study the e f f e c t of i n h i b i t o r on the light-induced accumulation of isoprenoid compounds (68); the e f f e c t on s t e r o l synthesis was





1

Figure 3. Radish seedlings (4 days old) grown i n the dark (upper part) or i n the l i g h t (lower part) i n the presence of mevinolin from onset of germination. Note the lack of l a t e r a l root growth at the high i n h i b i t o r concentration.

Figure 4. Mevinolin-induced growth i n h i b i t i o n of the main root of l i g h t grown radish seedlings. Mean values + SD from 30 to 50 plants per condition. (Reprinted with permission from Ref. 68. Copyright 1983 Physiologia Plantarum.) Fuller and Nes; Ecology and Metabolism of Plant Lipids ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

120


Table II.

Fresh/dry weight, s t e r o l content and accumulation of the l i g h t for 6 days in the presence of mevinolin 0


Plant Part, Treatment Cotyledons 0 0.25 0.625 2.5 5.0

Ο 0.25 0.625 2.5 5.0 a D c

Free Sterols (Ug/100 parts)

0

0.62 0.66 0.63 0.63 0.63

1640 1647 1569 1453 1091

μΜ Mevinolin μΜ Mevinolin μΜ Mevinolin μ Mevinolin μΜ Mevinolin

3.9 4.2 4.1 3.3 2.2

0.23 0.25 0.26 0.25 0.18

593 571 521 426 276

μΜ μΜ μΜ μΜ μΜ

2.8 2.5 2.2 1.8 1.3

0.15 0.14 0.12 0.11 0.09

522 416 274 200 112

Mevinolin Mevinolin Mevinolin Mevinolin Mevinolin

Hypocotyls

Roots

g DW (100 parts)

4.7 4.9 4.6 3.9 3.4

μΜ μΜ μΜ μΜ μΜ

0 0.25 0.625 2.5 5.0

g FU (100 parts)

0

0

Mevinolin Mevinolin Mevinolin Mevinolin Mevinolin

Mean values of three independent experiments 100 Cotyledon pairs analyzed per single experiment 100 Hypocotyls per analysis




t o t a l ubiquinone (Q-9 + Q-10) i n radish seedlings grown in


% of controls

Q-9 + Q-10 (ug/100 parts)

% of controls

100 97 86 77 39

% Q-9 of t o t a l Q-9 + Q-10

100 100 96 89 67

61.1 59.2 52.7 46.8 23.7

8.6 8.6 10.1 11.2 12.5

100 96 88 72 47

12.2 11.6 10.3 8.5 4.8

100 95 84 69 39

7.2 8.6 10.5 12.8 14.3

100 79 52 39 22

17.0 15.9 11.8 8.0 7.0

100 94 69 47 41

6.3 9.5 11.4 13.0 16.6


121


122


p a r a l l e l e d by an i n h i b i t i o n of elongation of hypocotyl segment growth. Total ubiquinone content was about 40% of the control at the maximal i n h i b i t o r concentration regardless of what part of the seedling was analyzed (Table II). However, in radish seedlings mevinolin treatment did cause a s h i f t towards the synthesis of Q-9 at the expense of Q-10, the predominant homologue i n radish (Table II) . Interestingly, only the fresh weight, but not the dry weight, of radish cotyledons was affected, whereas i n hypocotyls, and more c l e a r l y i n roots, fresh weight and dry weight were diminished upon mevinolin treatment (Table II). Even though a clear i n h i b i t i o n of s t e r o l accumulation in cotyledons by mevinolin can be achieved at higher concentrations as compared to the roots, there was no i n h i b i t i o n of chlorophyll and carotenoid biosynthesis under these conditions ((68) and Table III) . At low mevinolin concentrations chlorophyll a+b and carotenoid content was even enhanced as compared to untreated controls (Table III). The synthesis of other isopentenoid compounds of ρlastidic origin such as phylloquinone and plastoquinone appeared to be s l i g h t l y enhanced at low i n h i b i t o r concentrations and hardly inhibited even at 5 μΜ (Table III). α - T o c o p h e r o l (Table III), which was considered e a r l i e r to be synthesized i n the chloroplast as well as in the cytoplasm (90. 91), does not react l i k e sterols (Table II), which are c l e a r l y cytoplasmic products, but rather l i k e phylloquinone, known to be exclusively synthesized in the ρ l a s t i d (92, 93). This result agrees with recent findings (94), demonstrating the capacity for α-tocopherol synthesis in envelope membranes from spinach chloroplasts. It appears that s t e r o l s , as they are mainly affected, might be involved in physiological processes governing water uptake and c e l l elongation growth (cf. (95)). Evidently, the diminished synthesis of s t e r o l s , needed for biomembrane formation (28, 29). makes a strong contribution to the growth-retardant e f f e c t of mevinolin. The drastic i n h i b i t i o n of s t e r o l biosynthesis by mevinolin indicates HVA biosynthesis to be r a t e - l i m i t i n g , as has already been demonstrated i n the case of animal c e l l s (cf. 1, 3-7). This may also be true for ubiquinone, but apparently not for other compounds investigated here. Even though conclusive evidence i s available to indicate an independent MVA-synthesizing machinery being present in plant mitochondria (10, 14), possibly regulated d i f f e r e n t i a l l y from that assayed in the ER (96, 97.), HMG-CoA reductase a c t i v i t y i s c l e a r l y i n h i b i t e d , which led us to conclude that mevinolin can penetrate the mitochondrial envelope. Of course, we do not exclude the p o s s i b i l i t y that mitochondrial MVA u t i l i z a t i o n - and thereby ubiquinone biosynthesis - might a d d i t i o n a l l y be linked to cytoplasmic MVA-synthesis, depending on the need of the organelle for additional IPP units (cf. 72). The mevinolin-induced shift in the Q-pattern toward homologues containing shorter isopentenoid side chains (Table II) might r e f l e c t the a b i l i t y of mitochondria to adjust the usage of isopentenyl units to the available substrate inside or outside of the organelle, thereby maintaining a basic rate of synthesis needed for a functional respiratory electron transport. The s l i g h t increase of phylloquinone and, to a somewhat lower extent,



c

0

a

μΜ Mevinolin μΜ Mevinolin μΜ Mevinolin μΜ Mevinolin μΜ Mevinolin

0

-

830 830 840 770

-

124 118 120 113

-

4930 4670 4770 4470

740

100

3970

-

113 113 114 104

100

Carotenoids X+c (ug/100) (%)

Mean values of three independent experiments. 100 Cotyledon pairs analyzed per single experiment 100 Hypocotyls per analysis

0 0.25 0.625 2.5 5.0

0

Chlorophylls a+b (ug/100) (%)

14.0 16.8 19.0 15.5 13.5

158 137

-

154 157 192

-

100 120 135 113 98

2 .2 2..6 3 .1 3..5 2..1

21.,6 20..2

103 89

23,.9 26. .7 25..0

100 118 141 159 96

85

-90

100 112 104

Phylloquinone (ug/100) (%)

a

100 102 124

Plastoquinone (ug/100) (%)

E f f e c t of mevinolin on ρ l a s t i d i c p r e n y l l i p i d s and prenylquinones

μΜ Mevinolin μΜ Mevinolin μΜ Mevinolin μΜ Mevinolin μΜ Mevinolin μΜ Mevinolin

Hypocotyls

0 0.25 0.625 1.25 2.5 5.0

Cotyledons

Plant Part, Treatment

Table III.


252 259 268 _ 240 212

84

-95

100 103 106

α-Tocopherol (ug/100) (%)


124


of plastoquinone, upon mevinolin treatment at concentrations below 2.5 μΜ might be explained by an increased a v a i l a b i l i t y of acetate to be routed towards p l a s t i d i c isopentenoid biosynthesis. This would require that the acetate from the cytoplasm penetrates the chloroplast envelope. E f f e c t of mevinolin on isoprenoid synthesis i n primary leaves of wheat. Mevinolin can to some extent i n h i b i t the biosynthesis and accumulation of chlorophylls and carotenoids when i t s u f f i c i e n t l y penetrates the l e a f , as shown i n experiments with leaf segments of e t i o l a t e d wheat seedlings floated on a mevinolin s o l u t i o n of 2 mm thickness i n P e t r i dishes and then exposed to l i g h t . Despite the extreme i n v i t r o e f f i c a c y of mevinolin i n i n h i b i t i n g microsomal plant HMG-CoA reductase (see Table I ) , high concentrations were needed to a f f e c t the light-induced i n vivo formation of cartenoids and chlorophylls (Table I I ) . The chlorophyll b accumulation was i n h i b i t e d to a higher degree than that of chlorophyll a. This can be explained by assuming a preceeding formation of chlorophyll-protein complex containing chlorophyll a present i n reaction centers of photosystem I and I I followed by the formation of (under these experimental conditions where no supply of storage products from the seeds i s possible) rudimentary l i g h t harvesting complexes containing chlorophyll b (12, 98). The light-induced change i n percent composition of carotenoids ( e s p e c i a l l y the increase of β-carotene levels) appeared to be p a r t i a l l y blocked only at the higher mevinolin l e v e l . Because 6-carotene i s mainly located i n the photosynthetic reaction centers (99) and known to protect chlorophyll a molecules from photooxidation; t h i s lower β-carotene l e v e l may also account f o r the apparently diminished accumulation of chlorophylls. In contrast to pigments, the s t e r o l accumulation (defined as increase over dark control) i s completely blocked by the lower mevinolin concentration (Table IV). With primary leaves of wheat, incubated under comparable conditions but supplied with [^C]-acetate and [^Hl-mevalonate, precursors able to enter the isoprenoid pathway before or a f t e r the HMG-CoA reductase step, respectively, i t was shown (75, 76.) that mevinolin could completely prevent acetate incroporation into phytosterols. Incorporation of t r i t i u m from labeled MVA was unaffected. This e l i m i n a t i o n of [ C]-acetate incorporation into phytosterols was observed at a mevinolin concentration which had no e f f e c t on chlorophyll and carotenoid accumulation i n controls; v i r t u a l l y i d e n t i c a l accumulation i n bands of TLC-plates i d e n t i f e d as pheophytins or β-carotene was found f o r controls and mevinolin treatment (Bach & Nes, unpublished). The l i m i t e d a b i l i t y of mevinolin to prevent pigment accumulation i n chloroplasts favors the assumption that ρlastids contain t h e i r own independent enzyme system f o r MVA production. The p l a s t i d i c envelope i s apparently not at a l l or only poorly permeable to mevinolin. The a b i l i t y of p l a s t i d s to synthesize MVA has been questioned (70, 71). Our observations, together with i n v i t r o measurements of enzyme a c t i v i t y (16, 21), support the view that p l a s t i d s possess t h e i r own HMG-CoA reductase. 14




Table IV.

125


D i f f e r e n t i a l i n h i b i t i o n of s t e r o l accumulation and of the light-induced p r e n y l l i p i d accumulation at high mevinolin concentration i n excised 9-day-old e t i o l a t e d primary Wheat leaves during 18 h of continuous white light. P r e n y l l i p i d s i n yg per 40 leaf segments. Mean of 2 runs with SD < 10%. X = Xanthopylls; c = β-carotene; x+c = sum of carotenoids. Chlorophylls and carotenoids were separated by TLC (1) and determined photometrically.

Parameter

Initial Value

F i n a l value control

F i n a l value + Mevinolin (μια) 125

500

a) P r e n y l l i p i d content Chlorophyll a Chlorophyll b Ratio a/b Carotenoids Ratio x/c Ratio a+b/x+c Free desmethylsterols

0 0 134 29 230

273 24 11 165 7.1 1.8 302

134 6.6 20 114 11.3 1.2 232

72 1.5 48 90 19.4 0.8 229

3.3 18.6 17.8

12.4 17.7 11.2

8.1 17.8 13.1

4.9 17.0 15.2

58.2 3.7

54.8 3.9

57.3 3.7

59.5 3.4

b) % composition of carotenoids β-carotene Violaxanthin Antheraxanthin + Luteinepoxide Lutein Neoxanthin



126


E f f e c t of mevinolin on the growth and chemical composition of c e l l suspension cultures of Silybum marianum. In order to t e s t the i n h i b i t o r i n a system where e x t r a c e l l u l a r transport from the s i t e of a p p l i c a t i o n to the expected s i t e of action i s not an important f a c t o r , we used c e l l suspension cultures of Silybum marianum (100). Cell suspension cultures were i n i t i a t e d i n flasks containing growth medium supplemented with mevinolin at various concentrations. After 3 and 6 days the c e l l s were analyzed (Table V). The data demonstrate that the e f f e c t of mevinolin becomes more evident at a l a t e r stage of growth. Also, i n t h i s system the i n h i b i t i o n by mevinolin of isoprenoid synthesis follows a gradient: S t e r o l s > ubiquinones > plastoquinone ^ carotenoids « cholorophylls, thereby emphasizing the r e s u l t s obtained with radish and wheat seedlings. Besides an o v e r a l l i n h i b i t i o n of ubiquinone accumulation to about 50% (on the basis of dry weight), mevinolin induced a s h i f t i n the homologue pattern towards a shorter side-chain, e.g., the appearance of Q-8 at the expense of Q-10 (with Q-9 being the by f a r predominant Q-homologue formed i n Silybum marianum c e l l s ) . Ryder and Goad (101) have demonstrated using suspension cultures of sycamore (Acer pseudoplatanus L.) that i n the presence of 5 mg/1 compact i n the incorporation of [^C]- leucine and [^C]-acetate into 4-desmethylsterols was inhibited to 95% and 99%, respectively, while C-MVA incorporation was unaffected. However, s t e r o l synthesis from endogenous precursors, measured by incorporation of [Me-I^C]-methionine into the side chain, continued at a reduced rate f o r at least 6 h a f t e r a d d i t i o n of the i n h i b i t o r . This r e s u l t suggested the presence of a pool of precursors which was presumably gradually depleted by the process of sterol biosynthesis but could not be replenished from acetate i n the presence of compactin (101). Compactin was also revealed to i n h i b i t growth of c a l l u s cultures of tobacco (102). At 5 μΜ the average i n h i b i t i o n (fresh weight of c a l l u s ) was between 45 and 80%, at ΙΟμΜ around 95%. Cytokinins such as N -isopentenyladenine or k i n e t i n at concentrations from 0 to 1.0 and 0 to 5μΜ, respectively, could not s u b s t a n t i a l l y counteract the growth i n h i b i t i o n by compact i n (102). Mevinolin as w e l l as compactin were recently tested using expiants of Helianthus tuber ο sus (103). The e f f e c t s on growth parameters (fresh and dry weight) were w e l l w i t h i n the range determined with c e l l cultures of Silybum. 14

6

Aspects of secondary p h y s i o l o g i c a l responses of seedlings and c e l l cultures upon mevinolin treatment. Some observations that were made by the use of our radish growth system provide further support f o r the importance of MVA synthesis f o r normal development of seedlings. In e a r l i e r work (81) i t was documented that k i n e t i n , an a r t i f i c i a l cytokinin, induced i n radish seedlings a growth response comparable to mevinolin, e.g., shortened main roots. Cytokinins have been considered to be senescence regulation factors i n many plant systems. By measuring fast &nd slow fluorescence k i n e t i c s , i t was revealed that mevinolin can i n h i b i t radish seedling senescence, e.g., as indicated by the maintainence of a functional photosynthetic apparatus (68). Thus,



0 (control) 0.625 1.25 2.5 5.0 10.0

day 6:

0 (control) 0.625 1.25 2.5 5.0 10.0

day 3:

I n i t i a l value

400 340 300 260 140 110

277 290 310 247 173 180

11

100 85 75 65 35 28

100 105 110 89 63 65

(%)

38.0 34.4 23.3 21.9 19.3 16.4

55.6 50.0 41.0 32.8 24.3 23.3

364

100 90.5 61.3 57.6 50.8 43.2

100 89.3 73.7 59.0 43.7 41.9

(%)

mg dw mg protein 100 mg susp. g dw

3.8 2.9 2.0 1.3 0.8 0.3

3.4 3.0 1.7 1.2 1.1 0.9

2.9

100 76 53 34 21 8

100 88 50 35 33 26

(%)

mg s t e r o l s g dw

92.4 80.3 73.2 69.9 60.3 58.5

94.1 87.3 84.2 85.7 81.5 76.5

100 86.9 79.2 75.6 65.3 63.3

100 92.8 89.5 91.1 86.6 81.3

65.1 48.3 42.1 40.7 35.7 34.0

63.9 54.6 55.4 52.7 44.8 45.2

100 74.2 64.7 62.5 54.8 52.2

100 85.4 86.7 82.5 70.1 70.7

(%)

232 210 234 207 183 178

229 209 227 216 174 155

100 88 101 86 72 66

100 91 99 94 73 65

(%)

199

99.4

146. 9 (%)

ug PQ-9 g dw

ug carot. g dw

ug Chl.a+b g dw

E f f e c t of mevinolin on a c e l l suspension culture of Silybum marianum

μΜ Mevinolin

Table V.


216 204 194 155 116 105

211 203 192 173 144 133

57

100 94 89 71 54 49

100 96 89 82 67 53

(%)

ug t o t a l Q-n g dw

1 1

1 1 1

1 1 1 1 1 1

/

/

/

/

/

/

/

/

/

/

/

0.7 0.4

5.9 2.9 1.7

6 3 1.8 1.6 1.4 1.4

molar r a t i o Q~8 / Q-10

128


the root growth i n h i b i t i n g a f f e c t by c y t o k i n i n was perplexing since, i n p l a n t s , roots are regarded to be the main source of cytokinins (104, 105)» cytokinins are then transported to the shoot v i a the xylem (106). However, f o r the complete i n h i b i t i o n of,say, N^-isopentenyladenine or zeatin synthesis, concentrations of mevinolin might be required that are magnitudes higher than that needed to completely knock out de novo s t e r o l synthesis. Thus root growth may be i n h i b i t e d by a lack of s t e r o l synthesis i n the treated roots while the c y t o k i n i n synthesis continues i n d i c a t i n g d i f f e r e n t i a l i n h i b i t i o n by mevinolin of the accumulation of various end-products of the multibranched isoprenoid pathway. I t has been reported (84) e f f e c t of b r a s s i n o l i d e treatment on senescence of dark-maintained discs of Xanthium leaves was opposite to equimolar concentrations of k i n e t i n , promoting senescence (loss of chlorophyll) as compared to the c o n t r o l . I t may w e l l be that the synthesis of brassinolide-type s t e r o i d hormones requires the de novo synthesis of a phytosterol as has been suggested by work that has been done i n Dr. W. David Nes* lab at Berkeley (107) which supports the t h e s i s of a s c r i b i n g d i f f e r e n t f u n c t i o n a l or metabolic roles f o r d i f f e r e n t s t e r o l s by measuring indivudual turnover rates. In t h i s regard i t i s i n t e r e s t i n g to note that a p p l i c a t i o n of s t e r o i d hormones to e t i o l a t e d mung beans (88) resulted, i n the formation of longer main roots and i n a strongly increased number of side roots. In radish, at very high concentrations of mevinolin (> 1 mg per l i t e r ) the formation of l a t e r a l roots (see Figure 4) was completely prevented, an e f f e c t that could be observed over the f u l l c u l t i v a t i o n period of 10 days. Mevinolin may induce changes i n the endogenous phytohormone balance: a small amount of b i o l o g i c a l l y synthesized MVA might be s u f f i c i e n t f o r the supply of the isopentenyl derived moiety of isopentenyl-adenine or z e a t i n (-riboside?) or the isopentenylation of t-RNA but not for that of steroid phytohormones, thereby leading to a promotion of k i n e t i n - l i k e growth responses. The increased accumulation of anthocyanins i n hypocotyls of mevinolin-treated radish seedlings (69) c e r t a i n l y does not simply r e f l e c t the routing away of acetate u n i t s from s t e r o l s ( c f . Figure 1) but also r e f l e c t s a more general response of plants upon treatment with chemicals (95). I n h i b i t o r s (see also below) a f f e c t i n g l a t e r steps i n phytosterol synthesis seem to a f f e c t various other products of the isoprenoid pathway before and a f t e r squalene formation (95). Secondary e f f e c t s of such growth regulators l i k e d i s r u p t i o n of the f u n c t i o n a l i n t e g r i t y of the endoplasmic reticulum or the plasmalemma, thus leading to changes i n water and ion transport capacity (108) or i n t e r a c t i o n with isoprenoid c a r r i e r proteins, as suggested by Nés et a l . (95), may a d d i t i o n a l l y account for t h e i r mode of action. Since both s t e r o l s and GA3 can independently reverse the retardant action of these biocides on stem growth (109. 110) i t was suggested by Nés et a l . (95) that several end-products of the isoprenoid pathway may act independently on developmental processes, but produce the same end response. This emphasizes the more general concept of a shade-type or sun-type growth response of plants upon biocide


t n a t

t n e




129


treatment as proposed by Lichtenthaler (111). Treatment of yeast c e l l cultures with systemic fungicides such as c l o t r i m a z o l and triadimefon resulted i n an accumulation of l a n o s t e r o l that, i n a secondary process, proved to be a highly s p e c i f i c feedback i n h i b i t o r of HMG-CoA reductase a c t i v i t y w i t h i n the yeast c e l l s (23). Since there i s some evidence that plant HMG-CoA reductase a c t i v i t y might also be regulated by feedback mechanisms, as demonstrated with pea seedlings (13, 15), s i m i l a r e f f e c t s may account f o r the i n h i b i t o r y potency of several biocides which cause an accumulation of intermediary products of the brached isopentenoid pathway, a problem which needs further i n v e s t i g a t i o n . E f f e c t of i n h i b i t o r s a f f e c t i n g l a t e steps i n phytosterol synthesis on growth of radish seedlings. HMG-CoA reductase and enzymes catalyzing late steps of phytosterol synthesis are membrane-bound. I n c e l l s , membrane-bound enzymes w i t h i n a biosynthetic pathway, such as phytosterol synthesis, might be less abundant than soluble ones. I n a d d i t i o n , the methods of regulating t h e i r a c t i v i t y include membrane e f f e c t s such as induced changes i n the l i p i d composition i n the microspheres around various enzyme molecules. The r e s u l t s obtained with mevinolin support the view that HMG-Coa reductase plays a key r o l e a t l e a s t i n the coarse control of phytosterol synthesis. However, t h i s does not exclude the p o s s i b i l i t y that membrane-bound enzymes catalyzing later steps of phytosterol synthesis might be responsible f o r fine-tuning substrate flux. Therefore, s i t e - s p e c i f i c i n h i b i t o r s of such regulating enzymes should e x h i b i t i n s i t u , a c l e a r e f f e c t on growth. The compounds (Figure 5) tested i n the radish system are arranged to indicate some s t r u c t u r a l r e l a t i o n s h i p s . However, t h i s does not indicate per se what enzyme i s the target of these chemicals. I t appears reasonable to c l a s s i f y them according to the enzyme(s) i n h i b i t e d . Most of these compounds, i n p a r t i c u l a r the series of substituted imidazoles Clotrimazole, Miconazole and Bifonazole ( t y p i c a l systemic fungicides) are reported to i n t e r f e r e with the oxidative 14 α-desmethylation by binding nitrogen to the haem i r o n of cytochrome P-450 (112. 113. 114). I n susceptible fungi the accumulation of erogsterol precursors r e t a i n i n g the 14 α-methyl group may have l e d to t h e i r being unsatifactorily packed with the f a t t y a c y l chains of the phospholipids of the fungal membranes (115). This could have l e d to an a l t e r e d membrane f l u i d i t y (116) which then causes a decreased a c t i v i t y of the membrane-bound desaturase, r e s u l t i n g i n an increase of saturated f a t t y acids (115). This group of 14 a-desmethylation i n h i b i t o r s also includes T r i a r i m o l and Triadimefon. The 14 α-desmethylation involves several reactions, including reduction (28). Since the azasterol A 25822 B, a natural a n t i b i o t i c o r i g i n a l l y i s o l a t e d from Geotrichum flavobrunneum and characterized by Chamberlain e t a l . (117), was revealed to a f f e c t -reductase i n yeast thereby causing the accumulation of ignosterol (118), t h i s compound may be included i n the same group. I t has also been shown that i n bramble c e l l s , cultured i n the presence of A 25822 B, A » -sterols accumulated at the expense of A - s t e r o l s (119). Another group (U 18666 A, 8

14

5


130


HO"


0

Epiminolanosterol

SC 32561

H

HO 7-Deoxycholate

H5C2N-CH

H C 5

2

-CH

2

-0

U 18666 A

2

2.3- Epiminosqualene OH H5C2'

N -CH -CH -0-^-Ç-CH H^-Ct 2

2

CH

3

2

H5C2'

CH

3

Naftifine Triparanol (MER-29)

Ç)

C.^Q_CH -0-CH-O-C. 2

CI

CH

Miconazole

2

Ο

—

Q-f-O

-N Bilonozole

Clotrimazole OH

Cl-^-O-CH-C-tC^Hg

CI" 1

Ν /) -Ν Triadimefon

Figure 5.

Ο

ad * rN S^ N. a Triarimol

XH3

^ C H KL

CH

3

3

3

"NES"

Structures of s t e r o l synthesis inhibitors used and arranged to indicate some structural relationships.




Triparanol, epiminolanosterol i s comprised of compounds that a f f e c t the reduction of (120-122) or the Οχ-transfer to the Δ of l a n o s t e r o l and cycloartenol (123). A t h i r d group contains i n h i b i t o r s of oxidosqualene c y c l i z a t i o n (epiminosqualene (124. 125). U 18666 A (126, 127). "NES"). A known antimycotic agent, N a t i f i n e , was found to i n h i b i t fungal squalene epoxidase (128). At t h i s point, however, i t has to be noted that most of the compounds mentioned above are able to i n h i b i t more than one enzyme, including HMG-CoA reductase (113), depending on the concentrations used or organisms tested, i n p a r t i c u l a r i n algae and plant c e l l cultures (129, 130 and l i t e r a t u r e c i t e d t h e r e i n ) . Another drug, SC-32561, has been reported to prevent c h o l e s t e r o l ester accumulation and induction of HMG-CoA reductase i n mammalian t i s s u e cultures and intact animals, presumably through i n d i r e c t feedback regulation (131). Among those compounds being completely i n a c t i v e i n the radish root test was SC-32561, presumably i n d i c a t i n g that oxygenated s t e r o l s or t h e i r derivatives might not be as e f f i c i e n t as feedback regulators as they are i n mammalian systems ( f o r a recent review see r e f . (132)), even though conclusive data on t h i s topic are lacking. Iminolanosterol was also completely i n a c t i v e (data not shown), but t h i s i n a b i l i t y to i n h i b i t growth might be due to i t s low s o l u b i l i t y i n water, thereby r e f l e c t i n g the main l i m i t a t i o n of the radish root system which works best i f the compounds are a) r e a d i l y water-soluble or b) can be s o l u b i l i z e d and kept i n s o l u t i o n by the a i d of reasonable concentrations of emulsifying agents. Bifonazole (data not shown) was less active than Miconazole, a very hydrophobic compound, and t h i s was less i n h i b i t o r y than Clotrimazole, which induced a s i m i l a r growth reaction (73) as was found f o r Triadimefon; both caused a dwarfing response i n radish (111). U 18666 A, Triparanol, and "NES" (Figure 6) were comparably e f f e c t i v e growth i n h i b i t o r s with 10 mg per l i t e r being the c r i t i c a l concentration where no further root growth over the i n i t i a l value at day 3 of germination was observed. T r i a r i m o l reached t h i s l e v e l at about 50 mg per l i t e r and was more a c t i v e than N a f t i f i n e (75). Iminosqualene was i n h i b i t o r y at > 5 rag per l i t e r , a concentration that i s at the l i m i t of i t s s o l u b i l i t y . I n a d d i t i o n , the preparation used contained impurities (inner epimino groups) and was probably unstable at room temperature. The "best" agent - even though less e f f e c t i v e than mevinolin - was the n a t u r a l l y produced azasterol (A 25822 B) which reached the forementioned l e v e l between 1 and 5 mg per l i t e r (73.). The experiments give further evidence that inhibition of phytosterol synthesis can cause similar morphological changes i n radish, e. g., a decrease i n root elongation growth. However, one difference between the e f f e c t s of mevinolin as compared to that of a l l other compounds tested has to be noted: The complete lack of l a t e r a l root formation i n the presence of high concentrations of mevinolin was never observed with other chemicals. I t frequently appeared that l a t e r a l root growth was even stimulated as compared to control plants. This may i n d i c a t e that at least one further MVA-derivative, other than s t e r o l s , i s involved not only i n regulation of growth but, more b a s i c a l l y , i n 2 4


131



132


NES control 5ppm

10ppm 20ppm 50 ppm

3A567 10 days of germination

Figure 6. Growth i n h i b i t i o n of the main root of l i g h t cultivated radish seedlings by imminium salt ("NES"). Mean values + SD from 25 to 30 plants per condition.





133

c e l l cycle dynamics. The formation of l a t e r a l roots might require p a r t i c u l a r l y raeristematic i n i t i a l s w i t h i n the root cambium where c e l l s are r a p i d l y d i v i d i n g , and mevinolin, at concentrations higher than 1 mg per l i t e r , presumably i n h i b i t s c e l l d i v i s i o n through a r r e s t i n g the c e l l s w i t h i n the c e l l cycle as i t does i n mammalian c e l l s (133-136). I t should be mentioned that i n crown g a l l tumors caused by ARrobacterium high concentrations of isopentenyladenine are synthesized ( f o r l i t e r a t u r e see r e f . (137) and one gene product of the TI plasmid obviously i s coding f o r a dimethylallylphrophosphate transferase which i s involved i n the formation of isopentenyladenosine from 5*AMP (138, 139). I n t h i s t i s s u e the rate of c e l l d i v i s i o n i s high and requires an overproduction of cytokinins (and auxin (137, 140). I n a d d i t i o n to isopentenyl-derived cytokinins, d o l i c h o l s are involved i n the regulation of the c e l l cycle of eukaryotes as cofactors i n the glycosylation cycle of proteins (141) and i n c e l l wall biosynthesis (142). At high concentrations, mevinolin might a f f e c t t h e i r synthesis, thereby leading to an i n h i b i t i o n of regulatory events i n the c e l l cycle which are independent from s t e r o l synthesis. Conclusions and Outlook From our r e s u l t s and those of other groups, there i s strong evidence that the key-regulating r o l e of HMG CoA reductase seems not to be confined only to the animal kingdom, but can also be extended to plants and fungi. Since mammalian c e l l s contain fewer classes of isopentenoid compounds and even fewer d i f f e r e n t c e l l compartments, the regulation of mevalonate and isopentenoid biosynthesis a t the step committed by the HMG-CoA reductase reaction i s f a r more extensively studied and might not be so complex as compared to plant c e l l s . Nevertheless, the models of regulation v a l i d f o r mammalian systems might serve as a basis f o r undrstanding the features of regulation of mevalonate biosynthesis and substrate flow into various classes of isoprenoids p o s s i b l y synthesized w i t h i n d i f f e r e n t organelles of the plant c e l l (Figure 1). The possible interference of mevinolin with the synthesis of recently described brassinolide-type s t e r o i d phytohormones (see l i t e r a t u r e c i t e d above) might open new aspects f o r experimental designs to elucidate s t e r o i d phytohormone-dependent regulation processes i n p l a n t s . Moreover, the i n t e r a c t i o n of mevinolin with HMG-CoA reductase a c t i v i t y and p o s s i b l y with the balance of several growth hormones and how these phytohormones i n t e r a c t with the regulation of isprenoid synthesis at the enzyme l e v e l , may account f o r t y p i c a l growth responses of plants upon biocide treatment and needs further i n v e s t i g a t i o n . The s p e c i f i c i t y of the i n h i b i t i o n by mevinolin through i t s high a f f i n i t y f o r HMG-CoA reductase may also serve as a model to develop h i g h l y e f f e c t i v e and s p e c i f i c a r t i f i c i a l biocides and to stimulate research i n t h i s topic. For example, c i t r i n i n , another a n t i b i o t i c having a b i c y c l i c structure produced by Pénicillium citrinum has been shown to i n h i b i t s t e r o l biosynthesis (143) at the s i t e of acetoacetyl-CoA t h i o l a s e (EC 2.3.2.9) and HMG-CoA


134


reductase (144), but rather high concentrations were required. This compound was reported to i n h i b i t plant growth (145) which we suppose i s also a result of interference with HMG-CoA reductase activity. This i s further evidence, besides our studies using mevinolin, to propose HMG-CoA reductase to be a good enzymic target for b i o l o g i c a l l y active biocides. In any case, i n our attempt to shed new l i g h t on the physiological r o l e of mevalonate biosynthesis and metabolism in plants, mevinolin has proved to be a very suitable t o o l and can help to further elucidate the role of a functional isopentenoid and p r e n y l l i p i d pathway in the regulation of plant growth and development.


Acknowledgments Part of this work was supported by the Deutsche Forschungsgemeinschaft. We are indebted to Mrs. Ute Sieber for assistance i n the preparation of the manuscript. Legend of symbols GA, G i b b e r e l l i c a c i d ; HMG-COA, 3-hydroxy-3-methylglutaryl-coenzyme MVA, mevalonic acid

A;

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