Ecology and Metabolism of Plant Lipids - American Chemical Society

Chapter 2

Biosynthesis of Chloroplast Glycerolipids 1

Downloaded by UNIV OF MASSACHUSETTS AMHERST on June 2, 2018 | https://pubs.acs.org Publication Date: December 24, 1987 | doi: 10.1021/bk-1987-0325.ch002

J. B. Mudd, D. G. Bishop , J. Sanchez, K. F. Kleppinger-Sparace, S. A. Sparace, J. Andrews, and S. Thomas ARCO Plant Cell Research Institute, 6560 Trinity Court, Dublin, CA 94568

The glycerolipids of the chloroplast comprise mono galactosyldiacylglycerol (MGDG)*, digalactosyldiacyl glycerol (DGDG), sulfoquin-ovosyldiacylglycerol (SQDG), and phosphatidylglycerol (PG). PG is the only one of these found outside the chloroplast. While the synthesis of fatty acids is exclusively in the plastid of higher plants, the synthesis of the unique glycero lipids of the chloroplast requires contributions from the cytoplasmic compartment. The fatty acids synthesized in the plastid are exported to the cytoplasmic compartment as acyl- CoAs generated by enzymic activity of the outer membrane of the plastid envelope. These acyl- CoAs are utilized in the synthesis of phospholipids in the mitochondria and the endoplasmic reticulum. The fatty acid specificity in the synthesis of phosphatidyl choline (PC) is such that palmitate (16:0) is never found at the sn-2 position. Thus the predominant molecular species are sn1-18, sn2-18 and sn1-16, sn2-18. These molecular species of PC supply the diacylglycerol (DAG) moiety for synthesis of MGDG, DGDG, and SQDG in the plastid. In some cases ("18:3 plants") the DAG from PC is the sole supplier of DAG moieties to the three glycolipids. The mechanism of transfer of the DAG from PC to the chloroplast moiety is unknown. 1

*Abbreviations: ACP, a c y l c a r r i e r p r o t e i n ; APS, adenosine-5 phosphatosulfate; CDP-DG, c y t i d i n e d i p h o s p h a t e - d i a c y l g l y c e r o l ; DAG, d i a c y l g l y c e r o l ; DGDG, d i g a l a c t o s y l d i a c y l g l y c e r o l ; FdH2, ferredoxin (reduced); LPA, lysophosphatidic a c i d ; MGDG, monogalactosyldiacylglycerol; PA, phosphatidic a c i d ; PC, phosphat i d y l c h o l i n e ; PG, phosphatidylglycerol; PGP, phosphatidylglycerolphosphate; PSSO3", protein-S-sulfonate; SQDG, sulfoquinovosyld i a c y l g l y c e r o l ; UDP-SQ, uridinediphosphate-sulfoquinovose. 'Current address: CSIRO, Division of Food Research, North Ryde, New South Wales, Australia 0097-6156/87/0325-0010$06.00/0 © 1987 American Chemical Society

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

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Chloroplast Glycerolipids


DAG i s also synthesized i n the p l a s t i d , but i n t h i s case the f a t t y a c i d s p e c i f i c i t y i s such that 16 C acids are almost without exception found a t the sn-2 p o s i t i o n . Thus the predominant molecular species are snl-18, sn2-l6, and snl-16, sn2-l6. These molecular species are the sole s u p p l i e r o f DAG moieties i n the synthesis o f PG. In some cases ("16:3 plants") these molecular species contribute to the synthesis o f MDGD, DGDG and SQDG.

L i p i d biosynthesis i n c h l o r o p l a s t s has been extensively studied for 25 years. We now have a good understanding o f the synthesis of f a t t y acids and g l y c e r o l i p i d s . Whereas f a t t y a c i d synthesis i n higher plants i s l o c a l i z e d i n the p l a s t i d , the synthesis o f g l y c e r o l i p i d s o f the p l a s t i d s depends to a large degree on enzymes i n the cytoplasmic compartment. This review attempts to emphasize recent developments i n the study o f g l y c e r o l i p i d metabolism i n the c h l o r o p l a s t . Detailed current information may be found i n the recently published proceedings o f a symposium on Structure, Function and Metabolism of Plant L i p i d s (1). Fatty Acid Synthesis The studies on f a t t y a c i d synthesis i n higher plants over the l a s t 25 years have led to a consensus about the i n d i v i d u a l reactions and t h e i r l o c a l i z a t i o n i n the c e l l . This consensus i s that the enzyme system for f a t t y a c i d synthesis i s procaryotic i n nature, that i s the enzymes are soluble and separable, and that the system i s l o c a l i z e d e n t i r e l y i n the p l a s t i d . Thus the membranes o f the mitochondria, the endoplasmic reticulum, the plasmalemma, the tonoplast, the nuclear membrane, and the G o l g i apparatus a l l depend for t h e i r f a t t y a c i d components on the a c t i v i t i e s o f the p l a s t i d s . In o u t l i n e the reactions o f f a t t y a c i d synthesis may be summarized: acetate + ATP + CoASH acetylCoA + HCO3 acetylCoA + ACP malonylCoA + ACP acetylACP + malonylACP P-ketoacylACP + reduced nicotinamide P-hydroxyacylACP enoylACP + reduced nicotinamide +

A T P

-> —> —> -+ -> -> —• ->

acetylCoA malonylCoA acetylACP malonylACP P-ketoacylACP P-hydroxyacylACP enoylACP acylACP

[1] [2] [31 [4] [5] [6] [7] [8]

Although there i s general agreement about the o u t l i n e o f the synthetic r e a c t i o n s , some questions may be raised on s p e c i f i c issues, such as the o r i g i n o f the precursors f o r f a t t y a c i d synthesis and the o r i g i n o f the reductants used. Acetate has been used widely as a precursor f o r f a t t y a c i d synthesis by i s o l a t e d c h l o r o p l a s t s l a r g e l y as a matter o f convenience and economy. Several studies have attempted to determine whether acetate i s the p h y s i o l o g i c a l precursor.



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ECOLOGY AND METABOLISM OF PLANT LIPIDS

Roughan etal (2) compared acetate, pyruvate and malonate as p o t e n t i a l precursors and found that acetate was about three times better than pyruvate while malonate was not used a t a l l . Consistent with t h i s r e s u l t i s the report o f Kuhn etal Q ) who found that acetate concentration i n plant t i s s u e i s i n the order of mM and that acetate thiokinase i s l o c a l i z e d i n the p l a s t i d . Nevertheless, a l t e r n a t i v e substrates can not be e n t i r e l y excluded at t h i s stage. Schulze-Siebert etal (4) have reported that when c h l o r o p l a s t s are incubated with bicarbonate, pyruvate accumulates i n the c h l o r o p l a s t . Furthermore Williams and Randall (5) have reported that pyruvate dehydrogenase of pea c h l o r o p l a s t s has an a c t i v i t y o f 6-9 umol/h/mg c h l o r o p h y l l . I t i s therefore conceivable that the a c e t y l CoA used i n the f i r s t steps o f f a t t y a c i d synthesis i s derived from pyruvate. The question as to whether p h o t o s y n t h e t i c a l l y f i x e d carbon dioxide can d i r e c t l y give r i s e to precursors o f f a t t y a c i d synthesis has a l s o received considerable a t t e n t i o n . S t i t t and ap Rees (6) have reported that the pathway from 3-phosphglyeerie a c i d to pyruvate i s incomplete i n pea c h l o r o p l a s t s because o f the absence of phosphoglyceric a c i d mutase. This r e s u l t would suggest that the f i r s t product of photosynthesis leaves the c h l o r o p l a s t and the precursor o f f a t t y a c i d synthesis (acetate or pyruvate) i s eventually returned to the c h l o r o p l a s t . The r e s u l t of Schulze-Siebertetal (4) with spinach c h l o r o p l a s t s appears to be consistent with the presence o f the phosphoglyceric a c i d mutase i n these c h l o r o p l a s t s . Furthermore the r e s u l t s o f Journet and Douce (J) obtained by using p l a s t i d s from c a u l i f l o w e r i n f l o r e s c e n c e , i n d i c a t e that they are capable o f the conversion o f 3-phosphoglyceric a c i d to a c e t y l CoA. The studies o f the i n d i v i d u a l enzymes o f f a t t y a c i d synthesis i n higher plants has shown that the two reductive steps, 0-ketoacyl ACP reductase and enoyl ACP reductase have d i f f e r e n t cofactor requirements. As a r e s u l t the synthesis o f f a t t y acids depends on the a v a i l a b i l i t y o f both NADH and NADPH. While the p r o v i s i o n of NADPH can be a t t r i b u t e d to the photosynthetic r e a c t i o n s , the source o f NADH i n the c h l o r o p l a s t i s l e s s c e r t a i n . Takahama etal (8) have demonstrated that the content o f NADPH i n the c h l o r o p l a s t i s influenced by i l l u m i n a t i o n as expected, but there i s no such f l u c t u a t i o n o f the o x i d a t i o n s t a t e o f NAD/NADH. The production o f NADH to be u t i l i z e d i n f a t t y a c i d synthesis would therefore appear to depend on dark reactions. One p o s s i b i l i t y would be by the a c t i o n o f pyruvate dehydrogenase, which would generate not only the NADH required for reduction i n f a t t y a c i d synthesis but a l s o the precursor a c e t y l CoA. Most studies o f f a t t y a c i d synthesis by i s o l a t e d c h l o r o p l a s t s are made under photosynthetic c o n d i t i o n s . I l l u m i n a t i o n o f the c h l o r o p l a s t s generates ATP and reductant necessary f o r the incorporation o f acetate i n t o the f a t t y a c i d s . Other e f f e c t s o f i l l u m i n a t i o n may influence f a t t y a c i d synthesis. For example the pH and the magnesium ion concentration o f the stroma both r i s e when the c h l o r o p l a s t i s i l l u m i n a t e d . I t should be noted that non-photosynthetic p l a s t i d s are a l s o assumed to be the sole s i t e o f f a t t y a c i d synthesis and they must have sources



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Chloroplast Glycerolipids

13

of ATP and reductant a l t e r n a t i v e to photosynthetic mechanisms. Sauer and Heise (J) have addressed the problem of f a t t y a c i d synthesis by c h l o r o p l a s t s i n the dark. They have used the dihydroxyacetone phosphate s h u t t l e as f i r s t described by Werdan etal(]§), which depends on the conversion of DHAP to glyceraldehyde-3-phosphate which i s o x i d i z e d by GPDH, generating ATP and NADPH. Since DHAP i s taken i n t o the c h l o r o p l a s t by the phosphate t r a n s l o c a t o r i n exchange f o r phosphate i t was necessary to include phosphate as a component of the s h u t t l e to counteract the p o t e n t i a l decrease o f phosphate i n the stroma. The o r i g i n a l purpose of the DHAP s h u t t l e was to obtain carbon d i o x i d e incorporation i n t o 3-PGA i n the dark which required ATP but not reductant. I t was therefore necessary to r e o x i d i z e the NADPH generated by the o x i d a t i o n of phosphoglyceraldehyde otherwise the production o f ATP would have been l i m i t e d by the lack o f NADP. The r e o x i d a t i o n o f NADPH was accomplished by the a d d i t i o n o f OAA which was reduced to malate i n the stroma. In the experiments o f Sauer and Heise (2) a l l three components o f the DHAP s h u t t l e were necessary to observe f a t t y a c i d synthesis i n the dark. This i s rather puzzling since the OAA would be expected to d r a i n o f f NADPH which one would think i s required f o r the reductive steps of f a t t y a c i d synthesis. Perhaps the components o f the s h u t t l e have other e f f e c t s than those o u t l i n e d above. The r e s u l t s o f Browse etal(11) a l s o bear on the question o f f a t t y a c i d synthesis i n the dark. They have reported rates o f f a t t y a c i d synthesis by l e a f d i s c s o f spinach kept i n darkness which were 12-20% o f the rates i n the l i g h t . Sauer and Heise (2) have a l s o demonstrated that the synthesis o f f a t t y acids i n the dark i s stimulated when an ionophore i s used to increase the magnesium ion concentration i n the stroma. This r e s u l t i n d i c a t e s that the increase i n stroma concentration o f magnesium ion during i l l u m i n a t i o n i s favorable f o r f a t t y a c i d synthesis. The optimum pH f o r f a t t y a c i d synthesis i s a l s o achieved during i l l u m i n a t i o n of the chloroplast. Fatty Acid U t i l i z a t i o n The long chain f a t t y acids synthesized by the c h l o r o p l a s t system are i n the form o f ACP d e r i v a t i v e s . At any point i n the f a b r i c a t i o n o f the chain there are four things that can happen to the a c y l moiety: 1) the a c y l ACP can be u t i l i z e d i n another cycle o f elongation, 2) the a c y l chain can be t r a n s f e r r e d to glycerol-3-phosphate, 3) the a c y l moiety can be exported from the c h l o r o p l a s t to the cytoplasmic compartment, and 4) the a c y l ACP can be desaturated. (Figure 1 ). The f a t t y a c i d chain lengths synthesized by the c h l o r o p l a s t are p r i m a r i l y 16 and 18. This c l e a r l y i m p l i e s ' t h a t f o r a c y l ACP of chain lengths l e s s than 16 the predominant r e a c t i o n i s f u r t h e r elongation, and that for chain lengths o f 18, elongation i s r a r e . For 16:0 ACP,18:0 ACP, and 18:1 ACP, both t r a n s f e r to the cytoplasmic compartment and a c y l a t i o n o f glycerol-3-phosphate are d i s t i n c t p o s s i b i l i t i e s . The concentration o f ACP i n the stroma has been determined to be 8 uM [Ohlrogge etal (12)], so only small


ECOLOGY AND METABOLISM OF PLANT LIPIDS

14


cytoplasm

•

16:0 ACP

18:0 ACP

18:1 ACP

I©

I®

acylation of G-3-P

stroma

Figure 1. U t i l i z a t i o n of a c y l ACP. The a c y l ACP generated by the f a t t y a c i d synthesising system can be elongated (reaction ® ) , transferred to glycerol-3-phosphate (reaction

Ecology and Metabolism of Plant Lipids - American Chemical Society

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