Margaret A. Egli

Characterization of maize acetyl-coenzyme A carboxylase

Maize (Zea mays L.) leaf acetyl-CoA carboxylase (ACCase) was purified about 500-fold by ammonium sulfate fractionation and gel filtration and blue Sepharose affinity and anion-exchange chromatography. Most ACCase activity (85%) recovered from the anion-exchange column was found in a highly purified fraction (specific activity 5.5 [mu]mol acid-stable product min-1 mg-1) that consisted primarily of a single 227-kD biotinylated polypeptide. The fraction represented 29% of the original activity and was designated ACCase I. A second partially purified ACCase activity (ACCase II) eluted earlier during anion-exchange chromatography, contained a single biotinylated polypeptide of 219 kD, was poorly recognized by antiserum raised against the ACCase I polypeptide, and was less inhibited by the herbicides haloxyfop or sethoxydim than was ACCase I. ACCase I and II both utilized propionyl-CoA as substrate about 50% as effectively as acetyl-CoA, and neither utilized methylcrotonyl-CoA. Immunoprecipitation with antiserum and protein blotting of crude extracts of leaf, embryo, and endosperm tissue and suspension cells indicated that most ACCase activity in these tissues was immunologically similar and consisted of ACCase I. Only leaves contained significant amounts of the ACCase II polypeptide; however, no ACCase II polypeptide was found in isolated mesophyll chloroplasts. The ACCase I and II polypeptides appear to be subunits of distinct ACCase isoforms.

A maize acetyl-coenzyme A carboxylase cDNA sequence.

ACCase (EC 6.4.1.2) catalyzes synthesis of the malonylCOA required for subsequent synthesis of fatty acids and secondary metabolites in plants. ACCase activity is positively correlated with rates of fatty acid synthesis in both leaves and developing oil seeds, and it is likely to play a key regulatory role in plant lipid synthesis (Post-Beittenmiller et al., 1993). In maize (Zeu muys), most ACCase activity is associated with a high molecular weight, dimeric, MF, plastid-localized polypeptide that is sensitive to inhibition by aryloxyphenoxypropionate and cyclohexanedione herbicides (Egli et al., 1993). Complete coding sequences for MF ACCase polypeptides have been published for wheat (Gornicki et al., 1994) and for severa1 dicotyledons (Roesler et al., 1994; Schulte et al., 1994; Shorrosh e t al., 1994). Part ia1 m a i z e and rice ACCase cDNAs have also been reported (Ashton et al., 1994; T. Sasaki, unpublished data). Here we describe the complete coding sequence of a maize ACCase (Table I). Antiserum to the major maize ACCase polypeptide (ACCase I; Egli et al., 1993) was used to select potential maize ACCase cDNA clones from an oligo(dT)-primed Agtll expression library derived from A188 maize seedling leaf (Dr. Stephen Gantt, University of Minnesota). Plaques from 14 of 800,000 clones were strongly recognized by the antiserum. Four clones that contained 3.5to 5.4-kb inserts were partially sequenced and found to be identical. The remaining 5’ coding sequence was obtained by partia1 sequencing of a 15-kb genomic clone whose 3‘ end hybridized to an ACCase cDNA probe (nt 3900-5932). The corresponding cDNA sequence was obtained by three successive rounds of RT-PCR, using oligonucleotide primers based on genomic apparent exon (5’) and known cDNA (3’) sequences. PCR products corresponded to nt 1 to 240, 217 to 610, and 537 to 2094 of the final sequence and were cloned into PCR-script (Stratagene). The original 5.4-kb cDNA clone No. 18-5 and PCR products from at least three

Expression of the Acc1 Gene-Encoded Acetyl-Coenzyme A Carboxylase in Developing Maize (Zea mays L.) Kernels

A mutation (Acc1-S2) in the structural gene for maize (Zea mays L.) acetyl-coenzyme A carboxylase (ACCase) that significantly reduces sethoxydim inhibition of leaf ACCase activity was used to investigate the gene-enzyme relationship regulating ACCase activity during oil deposition in developing kernels. Mutant embryo and endosperm ACCase activities were more than 600-fold less sensitive to sethoxydim inhibition than ACCase in wild-type kernel tissues. Moreover, in vitro cultured mutant kernels developed normally in the presence of sethoxydim concentrations that inhibited wild-type kernel development. The results indicate that the Acc1-encoded ACCase accounts for the majority of ACCase activity in developing maize kernels, suggesting that Acc1-encoded ACCase functions not only during membrane biogenesis in leaves but is also the predominant form of ACCase involved in storage lipid biosynthesis in maize embryos.