The role of pyruvate dehydrogenase and acetyl-coenzyme A synthetase in fatty acid synthesis in developing Arabidopsis seeds

Abstract

Acetyl-coenzyme A (acetyl-CoA) formed within the plastid is the precursor for the biosynthesis of fatty acids and, through them, a range of important biomolecules. The source of acetyl-CoA in the plastid is not known, but two enzymes are thought to be involved: acetyl-CoA synthetase and plastidic pyruvate dehydrogenase. To determine the importance of these two enzymes in synthesizing acetyl-CoA during lipid accumulation in developing Arabidopsis seeds, we isolated cDNA clones for acetyl-CoA synthetase and for the ptE1alpha- and ptE1beta-subunits of plastidic pyruvate dehydrogenase. To our knowledge, this is the first reported acetyl-CoA synthetase sequence from a plant source. The Arabidopsis acetyl-CoA synthetase preprotein has a calculated mass of 76,678 D, an apparent plastid targeting sequence, and the mature protein is a monomer of 70 to 72 kD. During silique development, the spatial and temporal patterns of the ptE1beta mRNA level are very similar to those of the mRNAs for the plastidic heteromeric acetyl-CoA carboxylase subunits. The pattern of ptE1beta mRNA accumulation strongly correlates with the formation of lipid within the developing embryo. In contrast, the level of mRNA for acetyl-CoA synthetase does not correlate in time and space with lipid accumulation. The highest level of accumulation of the mRNA for acetyl-CoA synthetase during silique development is within the funiculus. These mRNA data suggest a predominant role for plastidic pyruvate dehydrogenase in acetyl-CoA formation during lipid synthesis in seeds.

Figure 1

Multiple sequence alignments for ACS proteins from Arabidopsis, yeast, and E. coli. The Arabidopsis ACS protein was aligned with the ACS1 and ACS2 proteins from yeast and the ACS protein from E. coli using the CLUSTAL W program (Thompson et al., 1994). The alignment was visualized using BOXSHADE (Kay Hoffman and Michael D. Baron; http://ulrec.3.unil.ch\software\BOX_form.html). White letters in a black field represent identical amino acids in two or more sequences. White letters in a gray field indicate conserved substitutions.

Figure 2

Immunoprecipitation of ACS activity by an antiserum against the transgenic putative ACS protein expressed in E. coli. Arabidopsis leaf extract was incubated for 60 min with the indicated concentration of preimmune or antiserum raised against the recombinant protein followed by a 60-min incubation with protein A agarose followed by centrifugation. The remaining ACS activity in the supernatant was then measured.

Figure 3

Gel filtration analysis of ACS from Arabidopsis. The 30% to 55% ammonium sulfate precipitate from an Arabidopsis leaf extract was resolved by gel filtration chromatography on a Superdex 220 HR 10–30 column equilibrated in buffer B. The numbers at the top of the figure are molecular mass standards for chromatography. The fractions were analyzed for ACS activity and for the presence of immunoreactive ACS protein. The immunoreactive bands shown at the bottom of the figure contain protein from two column fractions and are aligned with the corresponding fractions.

Figure 4

Southern-blot analysis of Arabidopsis DNA probed with the cDNA for ACS. Approximate molecular masses (in kb) are indicated.

Figure 5

Accumulation of the mRNAs for ACS and ptE1β in leaves, buds, flowers, and developing siliques (at the indicated DAF) determined by quantitative northern-blot analysis. The data presented are typical of the results from four independent experiments. White bars, Pyruvate dehydrogenase; black bars, ACS.

Figure 6

Cellular localization of the mRNA encoding ACS and ptE1β during development of Arabidopsis siliques and seeds. Tissue sections were hybridized with antisense RNA probes to detect acetyl-CoA synthetase mRNA (12-d exposures) (A, D, G, J, M, P, and T–X) and the ptE1β mRNA (4-d exposures) (B, E, H, K, N, and Q). The accumulation of the mRNA coding for the carboxyl-transferase-α-subunit of plastidic ACCase (4-d exposures) is shown for comparison (Ke et al., 2000) (C, F, I, L, O, and R). A single, typical control is shown in which sense ptE1β RNA is used as a probe (S). The control was processed exactly like the experimental sample. A to C, Siliques 1 DAF; D to F and T, siliques 3 DAF; G to I, siliques 5 DAF; J, K, L,and S, siliques 7 DAF; M to O, siliques 9 DAF; and P to R, siliques 12 DAF. Seeds at 1 (U), 2 (V), 3 (W), and 4 (X) d after imbibition probed for ACS mRNA. Tissue sections are stained with toluidine blue O. sc, Seed coat; rt, root; cot, cotyledon; w, silique wall; ii, inner integument of ovule; oi, outer integument of ovule; o, ovule; ge, globular embryo; he, heart embryo; te, torpedo embryo; ce, curled embryo; me, mature embryo; s, central septum; fu, funiculus. Bar = 55 μm in A through U;130 μm in V; 250 μm in W; 300 μm in X.

Figure 6

Cellular localization of the mRNA encoding ACS and ptE1β during development of Arabidopsis siliques and seeds. Tissue sections were hybridized with antisense RNA probes to detect acetyl-CoA synthetase mRNA (12-d exposures) (A, D, G, J, M, P, and T–X) and the ptE1β mRNA (4-d exposures) (B, E, H, K, N, and Q). The accumulation of the mRNA coding for the carboxyl-transferase-α-subunit of plastidic ACCase (4-d exposures) is shown for comparison (Ke et al., 2000) (C, F, I, L, O, and R). A single, typical control is shown in which sense ptE1β RNA is used as a probe (S). The control was processed exactly like the experimental sample. A to C, Siliques 1 DAF; D to F and T, siliques 3 DAF; G to I, siliques 5 DAF; J, K, L,and S, siliques 7 DAF; M to O, siliques 9 DAF; and P to R, siliques 12 DAF. Seeds at 1 (U), 2 (V), 3 (W), and 4 (X) d after imbibition probed for ACS mRNA. Tissue sections are stained with toluidine blue O. sc, Seed coat; rt, root; cot, cotyledon; w, silique wall; ii, inner integument of ovule; oi, outer integument of ovule; o, ovule; ge, globular embryo; he, heart embryo; te, torpedo embryo; ce, curled embryo; me, mature embryo; s, central septum; fu, funiculus. Bar = 55 μm in A through U;130 μm in V; 250 μm in W; 300 μm in X.