No abstract

The presence and the absence of a prokaryote type and a eukaryote type of acetyl-CoA carboxylase (EC 6.4.1.2; ACCase) were examined in members of 28 plant families by two distinct methods: the detection of biotinylated subunits of ACCase with a streptavidin probe, and the detection of the accD gene, which encodes a subunit of the prokaryotic ACCase, by Southern hybridization analysis. The protein extracts of all the plants studied contained a biotinylated polypeptide of 220 kDa, which was probably the eukaryotic ACCase. All the plants but those belonging to Gramineae also contained a biotinylated polypeptide of ca. 35 kDa, which is a putative subunit of the prokaryotic ACCase. In all plants but those in Gramineae, the ca. 35 kDa polypeptide was found in the protein extracts of plastids, while the 220 kDa polypeptide was absent from these plastid extracts. The plastid extracts of the plants in Gramineae contained the 220 kDa polypeptide, as did the homogenates of the leaves. Southern hybridization analysis demonstrated that all the plants but those in the Gramineae contained the accD gene. These findings suggest that most higher plants have the prokaryotic ACCase in the plastids and the eukaryotic ACCase in the cytosol. Only Gramineae plants might contain the eukaryotic ACCases both in the plastids and in the cytosol. The origin of the plastid-located eukaryotic ACCase in Gramineae is discussed as the first possible example of substitution of a plastid gene by a nuclear gene for a non-ribosomal component.

Acetyl-CoA carboxylase (ACCase, EC 6.4.1.2) catalyzes the synthesis of malonyl-CoA, the first intermediate in fatty acid synthesis. We studied the llization of two forms, the prokaryote and the eukaryote forms, of ACCase in pea leaves by comparing the blotin polypeptides of the two ACCases in protein extract from leaves and plastids.We found that the two forms of ACCase were in diferent cell compartments of pea leaves; the prokaryote form was in the plastids, and the eukaryote form was elsewhere, probably in the cytosol. This result suested the existence of two sites of malonyl-CoA thes. The Gramineae, such as rice and wheat, which lack the accD gene encoding one of the subunits ofthe prokaryote form ofACCase in their chloroplast genomes, did not have the prokaryote form of the enzyme but had the eukaryote form. The selectlve grass herbicides of the diphenoxyproponic acid type and the cyclohexanedione type, in vitro, inhibited plastidic ACCase of the eukaryote form from wheat but did not inhibit that of the prokaryote form from pea, suggeting that the origin of the tolerance of intact pea plant toward these herbicides is partly in the insensitivity of the prokaryote form of the enzyme. The origin of the susceptibility of the Gramineae plants toward these herbicides seems to lie in the presence of the herbicide-sensitive eukaryote form and the absence ofthe insenitive prokaryote form due to the lack of the accD gene in plastid.Acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) catalyzes the ATP-dependent carboxylation of acetyl-CoA to form malonyl-CoA. The reaction is the first committed step in the synthesis of fatty acid, providing the essential substrate for fatty acid synthesis. There are two forms of ACCase: a prokaryote form consisting of three protein components biotin carboxylase, carboxyltransferase, and biotin carboxylase carrier protein (1)-and a eukaryote form consisting of three functional domains on a single polypeptide (2-7). It has been believed that, in plants, plastids are the major site of fatty acid synthesis (8) and that only the eukaryote form ofthe enzyme in plastids participates in the synthesis (9, 10). Early studies reported the existence of the prokaryote form in the spinach chloroplast (11), but this finding has been dismissed because the prokaryote form has not yet been purified and the purified ACCases from various plants are all eukaryote form consisting of a subunit size of -200 kDa (5-7), like that of the mammal enzyme (2). ACCase gene encoding the 230-kDa polypeptide has been isolated from a photosynthetic eukaryote alga (12). This large polypeptide is probably nuclearencoded because this sequence is not found in the complete sequence ofchloroplast genomes (13-15). However, recently we have obtained evidence ofthe existence of the prokaryote form in pea chloroplasts by identifying one of the subunits of carboxyltransferase, the chloroplast-encoded accD protein (16). This finding supports the early studies and indicates the existence of two forms of ACCase in pea plants (16,17). ...

Fatty acid synthesis in chloroplasts is regulated by light. The synthesis of malonyl-CoA, which is catalyzed by acetyl-CoA carboxylase (ACCase) and is the first committed step, is modulated by light͞dark. Plants have ACCase in plastids and the cytosol. To determine the possible involvement of a redox cascade in light͞dark modulation of ACCase, the effect of DTT, a known reductant of S-S bonds, was examined in vitro for the partially purified ACCase from pea plant. Only the plastidic ACCase was activated by DTT. This enzyme was activated in vitro more efficiently by reduced thioredoxin, which is a transducer of redox potential during illumination, than by DTT alone. Chloroplast thioredoxin-f activated the enzyme more efficiently than thioredoxin-m. The ACCase also was activated by thioredoxin reduced enzymatically with NADPH and NADP-thioredoxin reductase. These findings suggest that the reduction of ACCase is needed for activation of the enzyme, and a redox potential generated by photosynthesis is involved in its activation through thioredoxin as for enzymes of the reductive pentose phosphate cycle. The catalytic activity of ACCase was maximum at pH 8 and 2-5 mM Mg 2؉ , indicating that light-produced changes in stromal pH and Mg 2؉ concentration modulate ACCase activity. These results suggest that light directly modulates a regulatory site of plastidic prokaryotic form of ACCase via a signal transduction pathway of a redox cascade and indirectly modulates its catalytic activity via stromal pH and Mg 2؉ concentration. A redox cascade is likely to link between light and fatty acid synthesis, resulting in coordination of fatty acid synthesis with photosynthesis.

Although the biochemical pathways for fatty acid synthesis are more or less similar in plants and animals (Harwood, 1988), there is a major cell biological difference between these two groups of eukaryotes. In plants, the major site of fatty acid synthesis is the plastid, an organelle absent from the animal cell. Many aspects of plastid biology, including fatty acid synthesis, reflect the organelle's origins as a prokaryotic symbiont. The synthesis of fatty acids, such as palmitic acid, the prototype 16-carbon fatty acid, requires one molecule of acetyl-COA and seven molecules of malonyl-COA, which are added sequentially with the addition of two carbons to the growing fatty acid chain and the release of CO, at each step. These reactions are catalyzed by fatty acid synthase, an enzyme complex known to exist in a prokaryotic and a eukaryotic form (Wakil et al., 1983;Harwood, 1988). The prokaryotic form (type 11) of fatty acid synthase is found in plants. The synthase is composed of severa1 dissociable proteins, whereas the eukaryotic form (type I) found in animals and yeasts is composed of one or two large multifunctional, nondissociable proteins. For either form, the synthesis requires malonyl-COA, which is supplied by ACCase in the following reaction:In plant cells, large amounts of malonyl-COA are needed in the plastids to sustain fatty acid synthesis, but malonyl-COA is also needed in the cytosol for the elongation of fatty acids exported from the plastids and for the synthesis of flavonoids and phytoalexins. As with fatty acid synthase, ACCase also occurs in prokaryotic and eukaryotic forms in nature. The prokaryotic form is composed of dissociable polypeptides, whereas the eukaryotic form is a homodimer of a multifunctional protein. But which form(s) of this enzyme occur(s) in plants? Both or only one? This biochemical mystery, which has been around since 1972, has finally been solved and the answer is intriguing, both from a ' Present address:

Acetyl-CoA carboxylase (ACCase) in plastids is a key enzyme regulating the rate of de novo fatty acid biosynthesis in plants. Plastidic ACCase is composed of three nuclear-encoded subunits and one plastid-encoded accD subunit. To boost ACCase levels, we examined whether overexpression of accD elevates ACCase production. Using homologous recombination, we replaced the promoter of the accD operon in the tobacco plastid genome with a plastid rRNA-operon (rrn) promoter that directs enhanced expression in photosynthetic and non-photosynthetic organs, and successfully raised the total ACCase levels in plastids. This result suggests that the level of the accD subunit is a determinant of ACCase levels, and that enzyme levels are in part controlled post-transcriptionally at the level of subunit assembly. The resultant transformants grew normally and the fatty acid content was significantly increased in leaves, but not significantly in seeds. However, the transformants displayed extended leaf longevity and a twofold increase of seed yield over the control value, which eventually almost doubled the fatty acid production per plant of the transformants relative to control and wild-type plants. These findings offer a potential method for raising plant productivity and oil production.

No abstract

RNA editing is an important post-transcriptional process in chloroplasts and is thought to be functionally significant. Here we show a requirement of RNA editing for a functional enzyme. In peas, acetyl-CoA carboxylase (ACCase), a key enzyme of fatty acid synthesis, is composed of biotin carboxylase with the biotin carboxyl carrier protein and carboxyltransferase (CT). CT is composed of the nuclear-encoded ␣ polypeptide and the chloroplast-encoded ␤ polypeptide in peas. One nucleotide of the ␤ polypeptide mRNA, which is edited in pea chloroplasts, converts the serine codon to the leucine codon. We show that this RNA editing is required for functional CT by comparing the unedited and edited recombinant enzymes. In plants not having a leucine codon at the same position, editing was shown to take place so as to create the leucine codon, indicating that editing is needed for in vivo CT activity and therefore for ACCase. To our knowledge, ACCase is an essential enzyme, suggesting that the chloroplast RNA editing is necessary for these plants.