Link between light and fatty acid synthesis: thioredoxin-linked reductive activation of plastidic acetyl-CoA carboxylase

Abstract

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 light/dark. Plants have ACCase in plastids and the cytosol. To determine the possible involvement of a redox cascade in light/dark 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 Mg2+, indicating that light-produced changes in stromal pH and Mg2+ 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 Mg2+ concentration. A redox cascade is likely to link between light and fatty acid synthesis, resulting in coordination of fatty acid synthesis with photosynthesis.

Figure 1

Effects of thiol reagents on the ACCase activity. (A) ACCase activity was measured in the indicated concentration of a thiol reagent as described in Materials and Methods. Plastidic ACCase from pea, continuous lines; cytosolic ACCase from pea, dashed line. β-SH, 2-mercaptoethanol; GSH, reduced form of glutathione. Lipoic acid was dissolved into 50 mM Tricine⋅KOH (pH 8) containing 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamide, and 5 mM ɛ-amino-n-caproic acid at 20 mM, and 0–7 μl of 20 mM solution was added to 13-μl reaction mixture. The activity without any thiol reagent was 11 nmol/20 min per mg of protein for pea plastidic ACCase and 210 nmol/20 min per mg of protein for pea cytosolic ACCase. The activity is relative to that without a thiol reagent, taken to be 1. Results are the means of duplicate determinations that differed less than 10%. (B) The effects of preincubation of plastidic ACCase with DTT on activity were examined. Changes with time are shown. After the enzyme was incubated at 30°C for 10 min with 2 mM DTT, the reaction was started by the addition of buffer, ATP, MgCl₂, KCl, acetyl-CoA, and NaH¹⁴CO₃. The final concentrations of these reagents were the same as above. The final concentration of DTT was 1 mM.

Figure 2

Effects of reduced thioredoxin on plastidic ACCase activity. (A) Pea plastidic ACCase was preincubated with various concentrations of thioredoxin (oxidized form) in the presence (○) and absence (•) of 50 μM DTT or reduced thioredoxin (□) at 30°C for 10 min, and the reaction was started as in Fig. 1B. For no DTT, thioredoxin (oxidized form) was added. (B) Pea plastidic ACCase was preincubated with various concentrations of DTT in the presence (○) and absence (•) of 32 μM thioredoxin at 30°C for 10 min, and treated as in A. The final concentration of DTT was indicated. The activity in the presence of thioredoxin was divided by that in its absence at various DTT concentration, and the value obtained was plotted as enhancement (□). (C) Various concentrations of E. coli thioredoxin (○), spinach thioredoxin-f (•) or -m (□) were incubated with 50 μM DTT at 20°C for 10 min to reduce thioredoxin, and then the pea plastidic ACCase was added. After the mixture was further incubated at 30°C for 10 min, the enzyme reaction was started as in Fig. 1B. The final concentrations of thioredoxins were indicated.

Figure 3

Effects of pH and Mg²⁺ on plastidic ACCase activity. (A) Pea plastidic ACCase was preincubated with 2 mM DTT at pH 8.0 at 30°C for 10 min, and then the reaction was started as in Fig. 1B except that different pHs were used. The final concentration of DTT was 1 mM. •, Mops⋅KOH buffer; ○, Tricine⋅KOH buffer. (B) After incubation of the enzyme with DTT as in A, the reaction was started as in Fig. 1B except that different concentrations of MgCl₂ and pH were used, with Mops⋅KOH buffer at pH 7.1 and Tricine⋅KOH buffer at pH 8.0.