Plant Cell Reports

Plant Cell Reports (1986) 5 : 1 9 - 2 2

© Springer-Verlag 1986

Monoterpene formation by leucoplasts of Citrofortunella mitis and Citrus unshiu Steps and conditions of biosynthesis Ginette Pauly, Lionel Belingheri, Anne Marpeau, and Michel Gleizes Laboratoire de Physiologie Cellulaire V6g6tale, U.A.C.N.R.S. n ° 568, Universit6 de Bordeaux I, Avenue des Facult6s, F-33405 Talence Cedex, France Received August 2, 1985 / Revised version received December 2, 1985 - Communicated by A. M. Boudet

ABSTRACT Leucoplasts of i m m a t u r e calamondin and s a t s u m a iruits were i n c u b a t e d with [ I - I ~ c ] isopentenyl pyrophosp h a t e under various conditions. Optimal incorporation of the t r a c e r into geranyl pyrophosphate and m o n o t e r p e n e hydrocarbons occurred in the p r e s e n c e of exogenous dimethylallyl pyrophosphate and Mn 2+ which was more e f f e c t i ve than Mg 2+. The d e p e n d e n c e of dimethylallyl pyrophosp h a t e showed t h a t about 10 moles were required for 1 mole ol isopentenyl pyrophosphate for the best recovery in m o n o t e r p e n e hydrocarbon biosynthesis. A t i m e - c o u r s e incorporation of isopentenyl pyrophosphate revealed t h a t the C10 hydrocarbon e l a b o r a t i o n was d e p e n d e n t on the geranyl pyrophosphate production and a t no t i m e neryl pyrophosphate was synthesized by leucoplasts. The amount of labelled farnesyl pyrophosphate was r a t h e r low w h a t e ver t h e conditions used in the e x p e r i m e n t s and sesquiterpene hydrocarbon biosynthesis was never observed. Abbreviations : DMAPP, dimethylallyl pyrophosphate ; FPP, farnesyl pyrophosphate ; GPP, geranyl pyrophosphat e ; IPP, isopentenyl pyrophosphate ; LPP, linalyl pyrophosphate ; NPP, neryl pyrophosphate. INTRODUCTION Isopentenyl pyrophosphate formed from a c e t a t e and m e v a l o n a t e is a key i n t e r m e d i a t e in the biosynthesis of terpenoid d e r i v a t i v e s in which a m u l t i e n z y m e system is implied (Banthorpe et al., 1972 ; Gleizes, 1976 ; Loomis and Croteau, 1980 ; Poulter and Rilling, 1981). In the first step, IPP is c o n v e r t e d into dimethylallyl pyrophosphate under the action of isomerase. The condensation of the two isoprene units (IPP + DMAPP) leading to geranyl pyrophosphate is ensured by p r e n y l t r a n s f e r a s e . From GPP, the synthesis of acyclic or cyclic m o n o t e r p e n e hydrocarbons involves the p r e s e n c e of carbocyclases with the loss of a proton and oI the pyrophosphate moiety. In the last ten years, the e n z y m e system responsible for the m o n o t e r pene hydrocarbon synthesis from the C l 0 i n t e r m e d i a t e s has been studied with partially purified c a r b o c y c l a s e s isolated from s u p e r n a t a n t s obtained with Sctgv& officinMis leaves (Croteau and Karp, 1976, 1979 ; Gambliel and Croteau, 1982) and Citrus Hmonam flavedo (Chayet et al., 1977 ; Rojas e t al., 1983). These c a r b o c g z l a s e s a r e able to form m o n o t e r p e n e hydrocarbons from geranyl pyrophosphate, neryl pyrophosphate and even linalyl pyrophosphate (Portilla e t al., i982). Recently, evidence was provided t h a t , from IPP, all t h e s e b i o s y n t h e t i c steps were performed by leucoplasts isolated from young calamondin (Cit~ofo~tunegA mitis) fruits (Gleizes et al., 1983). The major m o n o t e r p e n e synthesized was limonene and its f o r m a t i o n

Offprint requests to: G. Pauly

required the p r e s e n c e o* divalent cations. The present work reports the action of DMAPP and divalent cations on the elongation of the isoprene chain from IPP to CI0 i n t e r m e d i a t e s and m o n o t e r p e n e hydrocarbons in leucoplasts isolated from the peel of calamondin and s a t s u m a (Cit'guS ansh&) unripe fruits. It e s t a blishes t h a t GPP is the only C I 0 p r e c u r s o r in the two sources of organelles. MATERIAL AND METHODS Chemicals [I-I#C] isopentenyl pyrophosphate (1.96 GBq mmol - l ) was purchased from A m e r s h a m - F r a n c e , Versailles. Dimethylallyl pyrophosphate was prepared according to Holloway and Popjak (1967) and purified on a d i e t h y l a m i noethyl (DEAE) cellulose column Muted with ammonium I o r m a t e . Alkaline phosphatase was obtained from Sigma (St. Louis, Mo., USA). Plant material The young fruits used in the e x p e r i m e n t s were collected on plants of calamondin growing in the greenhouse of the University oi Bordeaux I and on t r e e s of satsuma growing in open air at the Station de R e c h e r c h e Agronomique de Corse, San Guiliano, 20230 San Nicolao. Leucoplast preparation The method of isolation of organelles was carried out as previously described (Gleizes e t al., 1983), e x c e p t that KCI was omitted from the extraction medium and the discontinuous sucrose gradient in tricine-NaOH 50 raM, pH 6.5, was enlarged at 6 layers (0.73, 0.92, 1.20, 1.25, 1.30 and t.50 M) to improve the purification of organelles. Isolated leucoplasts obtained from calamondin were collected at the interface t.20-1.25 M, whereas with satsuma the best recovery and the most effective purified leucoplasts banded at the interface 0.73-0.92 M. When i t was necessary, the leucoplasts drawn off the sucrose gradient were broken by fast freezing and thawing, which destroys the organelles. Enzymatic reaction The basic r e a c t i o n mixture c o n t a i n e d purified int a c t leucoplasts collected on the sucrose gradient in t r i c i ne-NaOH, pH 6.5 and [ I - I # C ] IPP (4.7 nmol, 9.25 kBq). To this mixture were added according to the situation considered, 100 pM DMAPP, 0.1 mM MnCI 2 or 3 mM MgCI 2. The iinal volume was I ml. Incubations were p e r f o r m e d at 28°C in glass stoppered tubes for 3 h and were stopped

20 Table 1. E f f e c t s of DMAPP, Mn 2+, Mg 2+ on the incorporation of [1-14C] IPP into dimethylallyl (DMA) alcohol, geraniol, [arnesol and m o n o t e r p e n e hydrocarbons (CIoHC) by leucoplasts of calamondin and satsuma. Alcohols were either e x t r a c t e d as free terpenols (F.T.) a f t e r incubation or released afterl~.nzymatic hydrolysis (E.H.) of the incubation medium. The incubation medium contained tricine-NaOH, pH 6.5,[1- ~C] IPP (4.7 pM, 9.25 kBq). Protein amounts were 80 IJg (calamondin) and 70 k~g (satsuma) per incubation. C o n c e n t r a t i o n s of added compounds were 100 I~M DMAPP, 0.t mM Mn 2+, 5 mM Mg z+. Final volume l ml. IPP incorporated DMA alcohol Calamondin Leucoplasts + + + + + +

IPP IPP IPP IPP IPP IPP

+ + + + +

DMAPP Mn 2+ DMAPP + Mn 2+ Mg 2+ DMAPP + Mg 2+

(nmol mg -1

Geraniol

h-l)

Farnesol

CIOHC

F.T.

E.H.

F.T.

E.H.

F.T.

E.H.

0.11 0.01 0.06 0.01 0.08 0.02

0.96 0.62 1.37 0.16 1.25 0.37

0 0 tr 0.i0 0 0.02

0 0.08 tr 1.00 0 0.10

0 0 0.01 0.01 tr 0.01

0 0 0.06 0.04 0.05 0.66

0 0 0.15 2.13 0.06 0.53

0.01 0 0.02 0 0 0

0.38 0.33 0.42 0.08 0.66 0.14

0 0 0 0.22 0 0.1t~

0 0.06 0 2.23 tr 2.66

0 0 0 0 0 0

0 0 tr 0.04 tr 0.06

0 0 0.06 2.57 0.01 0.91

Satsuma Leucoplasts + + + + + +

IPP IPP IPP IPP IPP IPP

+ + + + +

DMAPP Mn 2+ DMAPP + Mn 2+ Mg 2+ DMAPP + Mg 2+

tr, t r a c e s for amounts less than 0.01 nmol mg -1 h -1

by shaking the mixture with 1.5 ml pentane. The separation oi hydrocarbons and oxygenated compounds formed during incubation was carried out as previously described (Gleizes et al., 1983).

E n z y m a t i c hydrolysis

ring incubation. Nevertheless, at pH 6.57 the action of phosphatases was rather low with leucoplasts of calamondin and negligible with those of satsuma. The major part of the pyrophosphates remained in the incubation medium. They were recovered as their corresponding alcohols a f t e r e n z y m a t i c hydrolysis by alkaline phosphatase (Fig. 1).

A f t e r pentane e x t r a c t i o n , the incubation mixture was left 5 min under a strong N 2 s t r e a m to remove solvent traces. 0.5 ml of an alkaline phosphatase preparation (5 mg phosphatase in 5 ml of 0.1 M glycine buffer, p h i l 0 ) was added plus 5 mM MgCI 2 in the mixtures where Mg z+ was previously o m i t t e d . Enzymatic hydrolyses were carried out 2 h at 30°C and prenols released by alkaline phosphatase were e x t r a c t e d with 2 ml d i e t h y l e t h e r and an aliquot counted by liquid scintillation s p e c t r o m e t r y .

6

2 ! !

Protein d e t e r m i n a t i o n

I

Amounts of proteins per incubation were d e t e r mined according to Lowry et al. (1951).

I

Radio gas c h r o m a t o g r a p h y Analyses were carried out with a gas c h r o m a t o graph Intersmat IGC 121 FL coupled with a Nuclear Chicago proportional counter. A column of 10% of i r e e f a t t y acid phase on chromosorb W HP g0-100 (300 cm long, 0.63 cm outer diameter) was used with a t e m p e r a t u r e programme from 70 ° to 245 ° C at 3°C min -1 for m o n o t e r p e n e hydrocarbons and 100 ° to 245°C at 5°C min -I for oxygenated terpenes. Argon flow r a t e was 35 ml min - l . H y d r o c a r bons or alcohols were added as carriers to the d i f f e r e n t analyses.

RESULTS A N D DISCUSSION As already d e m o n s t r a t e d (Gleizes e t a l , 1983), isolated leucoplasts of calamondin incubated in the presence of [1-14C] IPP synthesized cyclic hydrocarbons (mainly limonene and to a lesser e x t e n t c~- and ~ 3 - p i n e n e ) and free terpenols. The l a t t e r are the products of phosphatases present in the plant tissues and released during the preparation of organelles (Loomis and Croteau, 1980 ; Block e t al., 1980). These phosphatases partly hydrolyse IPP used as s u b s t r a t e and pyrophosphates elaborated du-

cc 0 I,0 14J I--

15 TIME

30 (rnin }

Fig. I - Radio-gas liquid chromatogram on f r e e f a t t y acid phase column of alcohols released a f t e r e n z y m a t i c hydrolysis of the pyrophosphates elaborated during incubation by leucoplasts of satsuma. The upper tracing r e p r e s e n t s the flame-ionisation response of carrier alcohols. The lower tracing is radioactivity. 1, isopentenol ; 2, dimethylallyl alcohol ; 3~ linalool ; 4, nerol ; 5, geraniol ; 6, nerolidoI ; 7, Z-farnesol ; 8, E-farnesoI.

21

4 o I

[

O T--

K 3

U v

E Q.

<

C ~"

2

>-

2

>

O

I--

U
50 DMAPP

100 (HM)

Fig. 2 - E f f e c t s of DMAPP on GPP (O) and m o n o t e r p e n e hydrocarbons (e) biosynthesis by leucoplasts of calamondin. Each 1 ml incubation mixture c o n t a i n e d sucrose (1.23 M) in tricine-NaOH, ,oH 6.5 (50 raM), E l - l # c ] IPP (#.7 IJM, 9.25 kBq), 0.i mM Mn z+ and 75 IJg leucoplast protein. Incubation t i m e : 3 h. In Table 1 are shown the d i f f e r e n t conditions of incubation with [1-1#C] IPP. In the first case where leucoplasts of calamondin and s a t s u m a are i n c u b a t e d with IPP alone, only the isomerase is a c t i v e and the large a m o u n t of dimethylallyl alcohol obtained r e f l e c t s an effic i e n t synthesis of DMAPP. No o t h e r m e c h a n i s m implying p r e n y l t r a n s f e r a s e s and c a r b o c y c l a s e s i n t e r v e n e d under t h e se conditions. In c o n t r a s t , an excess of DMAPP (100 FM) added to the incubation medium produces a slight synthesis of GPP. However, t h e r e was no f o r m a t i o n of hydrocarbons indicating t h a t carbocyclases were not a c t i v a t e d in this condition w h a t e v e r t h e origin of leucoplasts (calamondin or satsuma). In the absence of DMAPP, the addition of divalent ions to the incubation medium a c t s both on p r e n y l t r a n s f e r a s e s and c a r b o c y c l a s e s but the synthesis of hydrocarbons is low and the situation is similar to the previous case in spite of t h e good a m o u n t of labelled DMAPP e l a b o r a t e d during incubation. It appears t h a t t h e a b s e n c e of exogenous DMAPP limits t h e p r e n y l t r a n s f e r a s e a c t i v i t y and, consequently, the synthesis of GPP, which appears to be a limiting f a c t o r . The small a m o u n t of GPP synthesized is a l m o s t e n t i r e l y used e i t h e r for t h e synthesis of m o n o t e r p e n e hydrocarbons, or for chain elongation towards the f o r m a t i o n of farnesyl pyrophosphate. The addition of exogenous DMAPP in the presence of Mn 2+ or Mg 2+ produces an increase of m o n o t e r p e n e hydrocarbon biosynthesis, indicating t h a t the r e a c t i o n catalyzed by the C10 p r e n y l t r a n s f e r a s e is no longer a r a t e limiting step for t h e f o r m a t i o n of GPP, t h e precursor of t h e s e hydrocarbons. The increase of CI5 products depends mainly on the p r e s e n c e of Mg 2+ ions, as r e p o r t e d for pren y l t r a n s f e r a s e s from o t h e r sources (Allen and Banthorpe, 19gl ; de la F u e n t e e t al., 19gl ; P e r e z e t al., 1983) ; whereas Mn 2+ ions are essential for cyclase a c t i v i t y (Chayet e t al., i977). There is also a d i f f e r e n c e b e t w e e n the source of leucoplasts, being those from s a t s u m a t h e most a c t i v e in the biosynthesis of GPP, both with Mn 2+ or Mg 2+ as divalent cation. These results could i n d i c a t e t h a t under our e x p e r i m e n t a l conditions t h e r e are also d i f f e r e n c e s b e t w e e n C I 0 and C15 p r e n y l t r a n s f e r a s e s in leucoplasts from d i f f e r e n t sources. C o n t r a r i l y to other classes of plastids (chloroplasts and chromoplasts) which i n c o r p o r a t e readily and largely IPP into prenyl lipids and higher terpenoids (Block

O

1

¢3
0 1

TIME

2

3

4

(hours)

Fig. 3 - Time-course incorporation of [ l - l # C ] IPP into GPP (O) and m o n o t e r p e n e hydrocarbons ( e ) by leucoplasts of calamondin. Each 0.5 ml incubation m i x t u r e c o n t a i n e d sucrose (i.23 M) in tricine-NaOH buffer, pH 6.5 (50 raM), [1-1~C] IPP (%# ~M, 9.25kBq), 100 I~M DMAPP, 0.1 mM Mn "+ and 35 yg of leucoplast protein. et aI., 1980 ; Kreuz and Kleinig, 198l ; Camara et al., 1992 ; Lf]tke-Brinkhaus et al., 1985) without addition of D M A P P , the leucoplasts require an exogenous supply of this C5 unit. This requirement showed by our ~n u~{o experiments confirms the existence of the metabolic pool of D M A P P suggested in plants producing volatile terpenes by ~n v~uo experiments made from different precursors and resulting in an a s y m m e t r i c labelling of the molecule of various C I 0 t e r p e n e s ( B a n t h o r p e and Turnbull, 1966 ; Banthorpe e t al., 1970 ; C r o t e a u and Loomi% 1972 ;Wuu and Baisted, 1973). In Fig. 2, the dependance of m o n o t e r p e n e hydrocarbon biosynthesis on DMAPP was e x a m i n e d with ]eucoplasts of calamondin. Optimal incorporation of [I-I#c] IPP was obtained with about 50 HM DMAPP, for #.7 HM IPP in the incubation medium, and this result is in good a g r e e m e n t with the remarks of Pou]ter and Rilling (19gl) which indicated an equilibrium position of 1 IPP to 5-10 DMAPP molecules. In the e x p e r i m e n t s r e p o r t e d in Table 1 and Fig. I, the leucoplasts were used as i n t a c t organelles directly aft e r they were collected on the sucrose g r a d i e n t (Gleizes e t al., 1983). In order to elucidate w h e t h e r molecules such as IPP and DMAPP could be channeled into these organelles, a comparison of [1-1#C] IPP incorporation was carried out with i n t a c t and broken organelles with d i f f e r e n t conc e n t r a t i o n s of DMAPP. The data r e p o r t e d in Table 2 show much the same results from i n t a c t and broken organelles indicating t h a t IPP and DMAPP may access to i n t a c t leucoplasts. These results a g r e e with those previously e s t a blished on the incorporation of IPP by i n t a c t spinach chloroplasts (Kreuz and Kleinig, 1981). All the e x p e r i m e n t s show the absolute requirem e n t for divalent cations for t h e biosynthesis of m o n o t e r pene hydrocarbons as well as for the biosynthesis of GPP and FPP (Table 1). Nevertheless, Mn 2+ ions were found much more e f f e c t i v e than Mg 2+ ions in the cyclase a c t i vity. These results agree with those obtained with p a r t i a l ly purified cyclases e x t r a c t e d from Sct~v& o{{~o_~ncz~ lea-

22 Table 2. Incorporation of [1-14C] IPP into geraniol released after enzymatic hydrolysis and monoterpene hydrocarbons (C10Hc)by intact (I) and broken (B) leucoplasts of calamondin with different concentrations of DMAPP. The incubation mixtures contained sucrose (1.23 M) in tricine-NaOH, pH 6.5, [1-14C] IPP (4.7 pM, 9.25kBq), 0.1mM MnC12 and 60 ~g leucoplast protein. Incubation t i m e : 3h.

Geraniol I

12.5 25 50

0.21 0.45 1.00

B

I

0.54 1.10 1.70

Phytochemistry 20 : 3%40. Chem Comnmun 177 : 8.

Banthorpe DV, Mann 3, Turnbull KW (1970) C, 2689.

B

0.57 1.15 1.55

ves (Croteau and Karp, 1976, 1979 ; Gambliel and Croteau, 1982, 1984) and Cit~tt8 gimonttm flavedo (Chayet et al., 1977 ; Portilla et al., 1982 ; Rojas et al., 1983). Several mechanisms have been postulated concerning the action of ions and it has been suggested (Rojas et al., 1983) that the binding of Mn 2+ ions to the pyrophosphate group of the substrate will make it a better leaving group for the formation of an enzyme-bound carbocation. Recent results from the same group indicate that the true subst r a t e for cyclase activity is the complex GPP-Mn2+or NPPMn2+(Chayet et al., 1984).

In all considered situations indicated in Table 1, the only CI0 pyrophosphate synthesized from IPP was GPP. Neryl pyrophosphate and linalyl pyrophosphate which could be eventual precursors of monoterpene hydrocarbons have been never detected after enzymatic hydrolysis of the incubation medium (Fig. 1). This observation demonstrates that from IPP the only intermediate precursor in monoterpene hydrocarbon synthesis is GPP preferentially to NPP and LPP. This fact is confirmed by a time-course incorporation of [1-14C] IPP by leucoplasts of calamondin shown in Fig. 3. The synthesis of monoterpene hydrocarbons is very fast and remains linear as long as GPP is elaborated by the organelles. When the amount of GPP decreases in the medium, the synthesis of hydrocarbons is strongly reduced and tends to a constant value. Table 1 shows also a low but non negligible synthesis of farnesyl pyrophosphate, particularly with the teucoplasts of Cit~ofo~tuneg~ miti~ in the presence of DMAPP and Mg 2+ , but FPP was never metabolized by the leucoplasts and the biosynthesis of sesquiterpene hydrocarbons did not occur in these organelles (Gleizes et al., 1983). In conclusion, the leucoplasts are able to ensure the chain elongation of the C 5 units to form ClO (GPP), C15 (FPP) and even C20 (geranylgeranyl pyrop/~osphate, Gleizes et al., unpublished results), but only geranyl pyrophosphate is metabolized, in the presence of exogenous dimethylallyl pyrophosphate and divalent cations, to monoterpene hydrocarbons by these highly specialized plastids.

We thank Dr. L.M. Perez, Universidad de Chile, Santiago, Chile, for helpful comment and discussion. M.R. Vogel, I.N.R.A., San Guiliano, 20230 San Nicolao, France, is gratefully acknowledged for the gift of satsuma young fruits.

Chem

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ACKNOWLEDGEMENTS

3 Chem Soc

Banthorpe DV, Charlwood BV, Francis M30 (1972) Rev 72 : 115-155.

Camara B, Bardat F, Moneger R (1982) 127 : 255-258.

C 10 Hc

0.18 0.43 0.95

Allen BE, Banthorpe DV (1981)

Banthorpe DV, Turnbull KW (1966)

Block MA, 3oyard 3, Douce R (1980) ta 631 : 210-219.

IPP incorporated (nmol mg -1 h -1)

DMAPP (pM)

REFERENCES

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