Medium chain acyl-ACP hydrolysis activities of developing oilseeds

Phytochemistry, Vol. 33, No. 6, pp. 1353-1356, Printed in Great Bntain.

MEDIUM

1993

003 l-9422/93 $6.00 + 0.00 @)1!4!33 Pergamon Press Ltd

CHAIN ACYL-ACP HYDROLYSIS DEVELOPING OILSEEDS H. Calgene

MAELOR

ACTIVITIES

OF

DAVIES

Inc., 1920 Fifth St., Davis, CA 95616, U.S.A.

(Received in revised form 19 January 1993)

Key Word Index-Umbellularia cal$ornica; Cinnamomum camphora; Lauraceae; Ulmus americana; Ulmaceae; Cocos nucifera; Palmae; acyl-ACP, fatty acids; thioesterase.

Abstract-Cell-free preparations from the seed tissues of three different species that accumulate medium chain triglycerides were examined for medium chain acyl-ACP hydrolysis. Activities with qualitatively appropriate medium chain length specificities were found, suggesting that a thioesterase has evolved in diverse plant families for the production of Cs, Ci,, and CiZ fatty acids. A pathway kinetic argument is presented to explain quantitative differences between the thioesterase specificities determined in vitro and the fatty acids accumulated in uiuo.

INTUODUCTION

Seeds of the native Californian plant Umbellularia calijknica (California bay) contrast with those of most higher ‘oilseed’ plants in their accumulation of reserve triacylglycerols containing predominantly C,, and CiZ acyl groups [l]. Studies in our laboratory have established that the fatty acid biosynthesis in developing bay seeds is switched from typical long chain (C,, and Cis) produo tion to 12:0 formation by the induction of a 12:0-ACP thioesterase [2,3]. We demonstrated this mechanism by expression of the medium chain thioesterase in higher plant species that normally make long chain triacylglycerols [4]; considerable quantities of 12 :0 were produced and incorporated into the oil of the transformants seeds c41. Despite this progress, many questions remain concerning the biosynthesis of such commercially important medium chain fatty acids. Most importantly, in view of the alleged difficulties in finding medium chain thioesterases [S], one may ask whether U. californica is unique in using such an enzyme to terminate fatty acid biosynthesis at medium chain lengths. There is also the question of how 10:0 is produced in bay embryos, given the considerable proportion of 10:0 in the triacylglycerols and our observation of surprisingly low thioesterase activity on lO:O-ACP [3]. The results from the present paper, show that species from other plant families that accumulate medium chain triacylglycerols also exhibit medium chain specific acyl-ACP hydrolysis activities in their seed tissues. These activities exhibit chain length specificities different from that of the bay enzyme and the specificities are qualitatively appropriate to the respective seed acyl compositions. Additionally, a theoretical argument is presented showing that pathway kinetics may account for quantitative differences between the thioesterase chain length

specificities and the resulting seed acyl compositions. This model suggests how the relatively low lO:O-ACP thioesterase activity observed in crude bay embryo preparations could account for the considerable 10:0 accumulation in this tissue. RESULTSAND DIsCUSSION Acyl-ACP hydrolysis activities and seed acyl compositions

Pollard et al. [3] were able to use a crude extract in order to demonstrate medium chain acyl-ACP hydrolysis activity in immature bay embryos with subsequent work with the purified enzyme confirming these results [2, 41. Proceeding on the assumption that crude extract hydrolytic specificities could be informative, other seed tissues were examined for signs of medium chain thioesterases (Fig. 1). In the following discussion the term ‘medium chain’ refers to the C&i, acyl series; interpretation of an activity on 14:0-ACP is difficult owing to the possibility that it may derive from either medium chain or long chain thioesterase enzymes. Medium chain acyl-ACP hydrolysis was observed in all cases. As with the bay preparation [3], these medium chain activities are typically lower than the activities on ‘long chain’ substrates (16:0-ACP and 18: l-ACP; Fig. 1). The latter are probably due to the action of the ubiquitous long chain acyl-ACP thioesterase (EC 3.1.2.14) [6]. Medium chain thioesterases may divert so much metabolite away from the fatty acid biosynthesis pathway that the pools of long chain acylACPs become very small. The long chain thioesterase activity would then have to be relatively high to ensure ongoing C,, and C,, fatty acid production. Medium chain length preferences of hydrolytic activities accord well with the medium chain acyl compositions of the respective tissues (Fig. 1). The most striking

1353

1354

H. M. 70 iii .ii 20 10 0 50 403 30 20

1 d

10

8 ;s

0

k

A 4-5 Jo; 20 g 10 0 60 50 40 30 20 10 0 Substrate acyl group

Fatty acid

Fig. 1. Acyl-ACP hydrolysis activities (left) and tissue fatty acyl compositions (right), of immature elm, camphor and bay embryos and coconut endosperm. Three plant families are represented, the Ulmaceae (elm), Palmae (coconut) and Lauraceae (camphor, bay). Bay data have been published previously [2,3] and are included here for reference. Medium chain hydrolysis activities were within the linear assay range, but with the exception of coconut all assays showed almost complete consumption of 18: l-ACP. Long chain thioesterase activities were therefore underestimated for elm, camphor and bay. A low activity of the bay preparation on fO:O-ACP was detectable using higher extract or substrate concentrations [3]. For each species the extraction and assays were performed three times with identical results (representative examples shown).

medium chain selectivity was observed with the elm preparation, the result being remarkably similar to that obtained with bay but with the most preferred substrate being lO:O-ACP instead of 12:0-ACP. Even the substrates that ‘flank’ the preferred one are utilized in the same order, i.e. just as the bay preparation has a preference for the 14:O substrate over the 10:0 one, so the elm preparation shows slight activity with 12:0-ACP and less with 8:0-ACP. These similarities suggest that elm and bay embryos have the same mechanism of medium chain production and provide the first evidence for this mechanism in a species outside the Lauraceae and for an activity preferring a chain length other than C,,. Coconut endosperm has been examined previously for the presence of medium chain acyl-ACP thioesterase, but both studies concluded that none was present [7,8]. The

DAVIES

apparent absence of thioesterase(s) to account for the considerable accumulation of 12:0 and other medium chains in this tissue contributed to much speculation about the underlying mechanism [S]. However, in the present investigation medium chain acyl-ACP hydrolysis activity was readily found (Fig. 1). This difference in detectability may be related to the developmental stage of the tissue and the method of extraction. Previous investigators imported immature coconuts and prepared acetone powders, whereas in the present work more mature, store-bought seeds were used and extracted directly. With coconut endosperm activity, the preferred substrate in the medium chain series is 12:0-ACP, again appropriately as this is the major acyl group in the reserve triacylglycerols. Significant action on the C, and C,, substrates was also observed (Fig. l), consistent with the less specific medium chain content of this tissue compared with bay and elm. Aithough a broadly specific medium chain activity has also been demonstrated in ~u~~eu species (Davies, H. M., Fan, C. and Hawkins, D. J.; Dormann, P., Spener, F. and Ohlrogge, J. B., unpublished), it is difficult to reconcile such an activity with the relatively narrow acyl compositions in such seeds. It should be noted that the relative enzyme activities on the different (medium) chain lengths are not the same as the relative amounts of the respective acyl groups accumulated in the tissue. This point is discussed below in reference to the Lauraceae. Additionally, it is worth noting that medium chain activities are higher in relation to long chain activities in coconut than in the other three species. Perhaps this difference is related to the lateness of medium chain deposition in coconut endosperm development [9]. It was of interest to examine another member of the Lauraceae for comparison with bay. Camphor embryos contain more 10:0 than those ofbay and interestingly this is reflected in the specificity of acyl-ACP hydrolysis in terms of a greater action on lO:O-ACP in relation to 12:0-ACP (Fig. 1). Basis of quantitative diflerence between hydrolytic spec$city and medium chain proportion in tissue

In camphor, bay and coconut the &at&e hydrol~ic activities on the different medium chain substrates do not correspond exactly to the relative proportions of acyi groups accumulated in the tissue. For example, in camphor the activity on lO:O-ACP is still less than that on 12:0-ACP, even though comparable amounts of 10:0 and 12:O are present in that tissue. This apparent mismatch has been considered in most detail with respect to the bay results, in which the activity on lO:O-ACP seems far too low relative to that on 12:0-ACP. One possible explanation would be the deposition of triacylglycerols of different fatty acyl compositions at different stages in embryo development. However, Davies et al. [2’J showed that bay embryos accumulate 10:0 and 12:0 in the same proportions throughout the period of triacylglycerol deposition, excluding the possibility of differential thioesterase expressions over time. The trivial possibility

Acyl-ACP hydrolysis activity of oilseeds that bay extracts contain an inhibitor of lO:O-ACP thioesterase has been tested by adding a small amount of elm extract to a bay preparation and assaying the elm lO:O-ACP hydrolysis activity in the mixture; the bay preparation had no adverse effect on the elm activity (data not shown). Many attempts have been made to increase the yield of lO:O-ACP hydrolysis activity in bay embryos, e.g. by extracting at different ionic strengths and pH values, and in the presence of protectants or detergents. None was effective in altering the proportions of the activities on Cl0 and C,, substrates (data not shown). Thus, it seems unlikely that there was a differential extraction of separate thioesterases. A more interesting possibility is that the products of the fatty acid biosynthesis pathway are determined not only by the substrate specificities of the individual enzymes but also by the overall pathway kinetics. This concept is readily demonstrated with a simple kinetic equivalent of the pathway diagram (Fig. 2). Qualitatively, this model depicts the simplest notion of medium chain thioesterase action in bay. Two medium chain enzymes are shown, one acting specifically on lO:O-ACP and one on 12:0ACP. This assumption is based on observations that crude and partially purified preparations did show measurable activity on lO:O-ACP when assayed at relatively high enzyme or substrate ~on~ent~tion~ whereas highly purified and clonally pure preparations did not [3, 41; it is possible that Davies et al. [2] purified the 12:0ACP thioesterase away from a small amount of 1O:O specific enzyme. The acyl-ACP substrates are available as free pools and the four steps of fatty acyl extension are treated as single reactions that supply 10: 0-ACP, convert lO:O-ACP to 12:0-ACP and extend the latter. All reactions are assumed to obey Michae~i~Men~n kinetics with respect to the substrates shown and kinetic constants are assigned arbitrarily to the extension reactions and thioesterases (Fig. 2). The concentrations of the two thioesterases may be altered to examine the impact of overall thioesterase specificity on the product composition, i.e. chain length specificity is achieved by blending the monospecific enzymes in different proportions. It

-

(fate=lO)

should be emphasized that because the model is only used to illustrate a concept, the various kinetic parameters need not, and do not, represent any measured values. However, it can be seen that realistic combinations of kinetic consents were used (e.g., the acyl extension kinetics are similar to the thioesterase kinetics, all K, terms are comparable, etc.). Results of kinetic simulations using this model are shown in Table 1. A constant influx of lO:O-ACP was supplied to the pathway and linear rates of 10: 0 and 12: 0 production were recorded when the respective acyl-ACP pool sizes had stabilized (i.e., at steady state). Progress curves for one of the simulations are shown in Fig. 3. The

Table 1. Simulation of the action of acyl-ACP thioesterases in fatty acid bios~thesis Product formation (rate)

Product composition (%)

Ratio of 12/10 thioesterases

to:0

12:o

1o:o

12:o

25.0* 125 6.3 3.1 1.6

5.2 6.8 8.1 9.0 9.5

4.7 3.2 1.9 1.0 0.5

52.5 68.3 81.2 89.9 94.7

47.5 31.7 18.8 10.1 5.4

The metabolic pathway model and associated kinetic constants are shown in Fig. 2 Simulations were performed by ‘Stelia’ software as described in Experimental. All units are arbitrary. Each simulation was begun from time zero (all acyl-ACP pools empty) and the linear rates of 10:0 and 12:0 product formation were recorded when a steady state had been reached, as defined by constant pool sizes of lO:O-ACP and 12:0-ACP. The ratio of thioesterase activities was varied solely by adjusting the quantity of lO:O-ACP thioesterase. *Progress curves shown in Fig. 2 correspond to this first simulation.

12:o Cl0

,

1355

t

Thioestaraos wy)

(1,2,7.5)

)

t

Cl2 l”hioe8tom1Iw C2sjW

W97.5)

Fig. 2. Hypoth~~l model of acyl-ACP thioesterase action in relation to fatty acid biosynthesis. A pool of lO:O-ACP is fed at a constant rate by the fatty acid biosynthesis pathway. The same pathway effects its conversion to 12:0-ACP and conversion of the latter to longer chain length acyl-ACPs, and ultimately to long chain fatty acids. These extension reactions are treated as single enzymes. Individual medium chain acyl-ACP thioesterases act on the respective acyl-ACP pools. All substrates other than those shown are assumed to be in excess. All reactions exhibit Michaelis-Menten kinetics, the numbers in parentheses indicating relative enzyme concentration, XI, and V,,,.

Fig. 3. Typical kinetic simulation of acyi-ACP thioesterase action in relation to fatty acid biosynthesis. Kinetic parameters were as shown in Fig. 2 and in the first line of Table 1. The simulation was started with all intermediate and product pools empty. T’he pools of fO:O-ACP and 12%ACP stabilized at constant levels early in the simulation (by 2.5 time units), after which time 10: 0,12: 0 and long chain products were produced at constant rates. This is the steady-state situation used to obtain the data for Table 1.

1356

H. M. DAVIES

concentrations of the 12:0 specific thioesterase and all the kinetic parameters were kept constant while the amount of the 10:0 specific activity was altered (Table 1). The requirement that long chain fatty acids should continue to be made at a low rate, as observed in developing bay embryos [3], was satisfied by the five simulations shown (e.g. Fig. 3). When the two medium chain thioesterase concentrations were nearly equal (12:0/10:0 thioesterase concentration ratio 1.6), the medium chain products comprised almost entirely lO:O, indicating the considerable impact of the first thioesterase on flux through the extension pathway. Lowering the amount of lO:OACP thioesterase reduced the proportion of 10:0 in the product, as expected. However, this effect was markedly non-linear; when the thioesterase acting on 10: 0 had been reduced to 4% of that on 12:0,10:0 still represented 53% of the products (Table 1). Note that in crude bay preparations the thioesterase action on lO:O-ACP is typically l-6% of that on 12:0-ACP [3, and unpublished observations], and in viuo 33% of the medium chain fatty acids accumulated are 10:O. Thus, the proportions of lO:O-ACP and 12: 0-ACP thioesterases in an extract would seem to be inappropriate and yet, theoretically, account for the observed proportions of products in vivo. EXPERIMENTAL Plant material. Immature seeds of California bay (Umbellularia californiea Nutt.), camphor (Cinnamomum camphora L.) and elm (Ulmus americana L.) were collected

locally from wild (bay) or ornamental specimen (camphor, elm) trees. With ref. to the published time course of seed development [2] the bay seeds were harvested at day 55. Camphor seeds were harvested when the gelatinous contents had just been replaced by embryonic tissue (early June), and the elm seeds when the embryo could readily be dissected from the green stage (early April). Embryos were dissected out as quickly as possible after collection, frozen immediately in liquid N, and stored at -70”. Coconuts (near-mature, ‘green’ seeds of Cocos nucifera L.) were purchased at local supermarket stores and the endosperm tissue was frozen and stored similarly. Preparation of crude extracts for enzyme assays. Each frozen tissue was powdered in a mortar and pestle under liquid N, prior to extraction. All subsequent steps were undertaken at &4”. The powdered embryo tissue was ground in a mortar and pestle with a small quantity of sand and extraction buffer (4 ml g-i fr. wt) comprising (for all species except coconut) 50 mM NaH,PO,-NaOH pH 7.5, 2 mM dithiothreitol (DTT), 2 mM Na ascorbate and 20% (v/v) glycerol. In addition, the elm extraction buffer contained 1% (w/v) PVP-10. The coconut extn buffer comprised 20 mM NaH,PO,-NaOH pH 8, 20 mM DTT, 20% (v/v) glycerol and 5% (w/v) PVP-10. The homogenate was centrifuged for 5 min at ca 10 000 g to provide a supernatant fr. (crude extract). Acyl-ACP hydrolysis assays. Acyl-radiolabelled acylACP substrates were prepd as described previously, with sp. act. of 1.98-2.22 GBq mmol-’ [3]. Assays were undertaken according to ref. [2] with final acyl-ACP

concns of 0.5 PM. The 100 ,~l assay system contained the following quantities of pre-diluted (with assay buffer) crude extracts: bay, 25 ~1 of a 15-fold dilution; camphor, 25 ~1 of a 20-fold dilution; coconut, 40 ~1 undiluted; elm, 25 ~1 of a 40-fold dilution. These quantities of extract were used to ensure that medium chain hydrolysis activities fell within the range of assay linearity. Activities were calculated from the amount of radioactive fatty acid product extracted by Et,O. Fatty acid compositions. Fatty acid analyses were performed on the powdered, immature embryo samples that had been used for the enzyme assays. Total acyl compositions were determined by transmethylation and GC essentially according to ref. [lo]. Simulation of pathway kinetics. The pathway model for acyl-ACP thioesterase action in fatty acid biosynthesis was constructed and operated using Stella flux modelling software (High Performance Systems) running on a Macintosh SE/30 computer. Software settings included computation by the Euler procedure and a simulation step time of 0.01. All results were verified to be free of computational artefacts by ensuring that none of the computed variables exhibited oscillations and by showing that exactly the same results were obtained when shorter step times were used. The application of this software to the study of metabolism and physiology has been reviewed [l 11. Acknowledgements-Dr Mike Pollard (currently at Agrigenetics Company, 5649 E. Buckeye Road, Madison, WI 53716) drew our attention to local camphor trees, Debbie Hawkins obtained coconuts from local stores, Eric Svee prepared the acyl-ACP substrates and Johanna DiMento and Cheryl Eriqat performed fatty acid analyses.

REFERENCES

Litchfield, C., Miller, E., Harlow, R. D. and Reiser, R. (1967) Lipids 2, 345. Davies, H. M., Anderson, L., Fan, C. and Hawkins, D. J. (1991) Arch. Biochem. Biophys. 298, 37. Pollard, M. R., Anderson, L., Fan, C., Hawkins, D. J. and Davies, H. M. (1991) Arch. Biochem. Biophys. 284, 306.

4. Voelker, T. A., Worrell, A., Anderson, L., Bleibaum, J., Fan, C., Hawkins, D. J., Radke, S. E. and Davies, H. M. (1992) Science 257, 72. 5. Harwood, J. L. (1988)A. Rev. Plant Physiol. Mo6ec. Biol. 39, 101. 6. Knutzon, D. S., Bleibaum, J. L., Nelsen, J., Kridl, J. C. and Thompson, G. A. (1992) Plant Physiol. (in press). 7. Ohlrogge, J. B., Shine, W. E. and Stumpf, P. K. (1978) Arch. Biochem. Biophys. 189,382. 8. 00, K. C. and Stumpf, P. K. (1979) Lipids 14, 132. 9. Padua-Resurrection, A. B. and Banzon, J. A. (1979) Phil. J. Coca. Stud. 4, 1. 10. Browse, J., McCourt, P. J. and Somerville, C. R. (1986) Analyt. Biochem. 152, 141. 11. Bogen, D. K. (1989) Science 246, 138.