Link between light and fatty acid synthesis: Thioredoxin-linked reductive activation of plastidic acetyl-CoA carboxylase (coordination with photosynthesisymalonyl-CoA synthesisypeayredox cascadeysignal transduction)

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 lightydark. Plants have ACCase in plastids and the cytosol. To determine the possible involvement of a redox cascade in lightydark 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 Mg21, indicating that light-produced changes in stromal pH and Mg21 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 Mg21 concentration. A redox cascade is likely to link between light and fatty acid synthesis, resulting in coordination of fatty acid synthesis with photosynthesis. Acetyl-CoA carboxylase (ACCase) regulates the rate of fatty acid synthesis in yeasts, animals, Escherichia coli, and plants (1, 2). In yeasts and animals, this enzyme activity is controlled by a variety of metabolites and by phosphorylation and dephosphorylation so that excess energy is stored in the form of fatty acids in response to the environmental conditions (1, 3). Such regulation has not yet been found in plants (4). Plant fatty acids are synthesized mainly in plastids, and a regulatory system different from that of other eukaryotes and characteristic of plastids may be involved. Regulation via redox is an important mechanism characteristic of chloroplasts (5). Enzyme activities of the reductive pentose phosphate cycle in chloroplasts are regulated via a redox cascade generated by light (5–9). In the light, electrons from photosystem I are shuttled through the electron transport chain to ferredoxin and are transferred to thioredoxins by ferredoxin-thioredoxin reductase, and then to target enzymes, reducing a disulfide bond(s) of the enzymes and changing their catalytic activities. Synthesis of certain proteins in chloroplasts also is regulated via a redox cascade (10). In the redox cascade, thioredoxin acts as a transducer of redox potential generated by the light reactions of photosynthesis, providing the chloroplasts with a mechanism to coordinate the activity of various components of photosynthesis to the presence or absence of light (5–9). De novo fatty acid synthesis in chloroplasts increases in the light and decreases in the dark. The first committed step of this synthesis, catalyzed by ACCase, is the formation of malonylCoA. Isolated chloroplasts incorporate acetate into malonylCoA within minutes when exposed to light and the incorporation decreases when exposure ends (11). This pattern of change has been partly explained by changes in ACCase activity via the pH, Mg21, and adenine nucleotide levels of the chloroplast stroma (2, 11–13). Fatty acid synthesis also may be regulated through a redox cascade, but evidence is lacking. Recently we identified two forms of ACCase and showed that most plants examined so far have the prokaryotic form in plastids and the eukaryotic form in cytosol. Of the plants examined, only Gramineae have the eukaryotic form in both plastids and the cytosol (14–17). The eukaryotic form of the enzyme from several plants has been purified and characterized, but the prokaryotic form has not. Here we obtained the prokaryotic form of the enzyme partially purified from pea and examined its properties to find if a redox cascade was involved in malonyl-CoA synthesis. We examined the effects of DTT and reduced thioredoxin on plastidic prokaryotic ACCase activity, in addition to the effects of pH and Mg21, and compared the effects with those on the eukaryotic form of ACCase. MATERIALS AND METHODS Reagents. Thioredoxin from E. coli and the reduced form of DL-a-lipoic acid (DL-6,8 thioctic acid) were purchased from Sigma. The reduced form of E. coli thioredoxin was generally prepared by the incubation of 0.32 mM thioredoxin in 50 mM TricinezKOH, pH 8, with 4 mM DTT at 30°C for 10 min, and then DTT was removed by centrifuging in a gel filtration column (Biospin 6, Bio-Rad). Recombinant spinach thioredoxins-f and -m, gifts from P. Schürmann (Université de Neuchâtel, Switzerland), were prepared as described (18). Thioredoxin reductase from E. coli was purchased from American Diagnostica (Greenwich, CT). Partial Purification of Plastidic ACCase. Pea plants (Pisum sativum cv. Alaska) were grown with a cycle of 16 hr of light and 8 hr of dark. Intact chloroplasts were isolated from leaves 9 days old with a Percoll gradient. The isolated chloroplasts were ruptured by a lysis buffer (50 mM TricinezKOH, pH 8y1 mM EDTAy10 mM 2-mercaptoethanoly1 mM phenylmethylThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y9411096-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: ACCase, acetyl-CoA carboxylase; BC, biotin carboxylase. *To whom reprint requests should be addressed. e-mail: sasaki@ nuagrl.agr.nagoya-u.ac.jp. †Present address: Ciba Speciality Chemicals Consumer Care Chemicals Division, 10-66 Miyuki-cho, Takarazuka 665, Japan.