A 233kDa subunit of acetyl-CoA carboxylase is encoded by the Acc1 gene

--Margaret A. Egli, Burle G. Gengenbach, John W. Gronwald1, David A. Somers and Donald L. Wyse

1USDA-ARS

Acetyl-CoA carboxylase (ACCase) is a biotinylated enzyme that catalyzes the formation of malonyl-CoA from acetyl-CoA and bicarbonate and is inhibited by several herbicides. Tissue culture selection for herbicide tolerance revealed that a partially dominant mutation in an ACCase gene (Acc1) of a selected maize line (S2) confers tolerance to sethoxydim and haloxyfop herbicides at both the whole plant and ACCase activity levels (Parker et al., PNAS 87:7175, 1990). Our eventual goal is to isolate the Acc1 gene from wildtype maize to better understand its expression and regulation during development of wildtype and mutant maize and to determine its role in fatty acid biosynthesis.

We previously described the purification of a high molecular weight ACCase from maize inbred A619 and the production of ACCase antiserum (Egli, MA et al., MNL 65:95, 1991). The possible existence of ACCase isozymes was suggested by the appearance of two high molecular weight, biotinylated polypeptides on Western blots of crude extracts from inbred leaves (MNL 65:95) and by a report of multiple ACCase activities in hybrid maize (Howard and Ridley, FEBS Lett. 261:261, 1990). We now report that (i) maize leaves contain two ACCase activities that differ in subunit size and sensitivity to herbicide inhibition and (ii) the Acc1 gene encodes a 233kDa polypeptide.

ACCase was purified from inbred B73 or mutant S2 seedling leaves by ammonium sulfate fractionation, gel filtration (S-400), affinity chromatography (Blue Sepharose), and anion exchange (Mono-Q). The ACCase activity elution profile from Mono-Q was the same for B73 and S2. Two peaks were eluted at approximately 210 (peak I) and 250mM KCl (peak II). Peak II was identical to the purified ACCase fraction that was previously used for antibody production and comprised approximately 85% of the total ACCase activity eluting from the column. Peaks I and II each contained a single biotinylated polypeptide of 225 and 233kDa, respectively. Molecular weights were estimated from 6% acrylamide gels that allowed a better separation than higher percent gels used previously. The 225kDa polypeptide was unlikely to be a proteolytic product of the 233kDa polypeptide because both were found in rapidly prepared crude extracts containing PMSF plus five other protease inhibitors.

Besides ACCase, other biotinylated carboxylases that utilize the substrates propionyl-CoA, methylcrotonyl-CoA, and pyruvate have been reported in plants. However, animal studies indicate subunit sizes of 70 to 125kDa for these carboxylases (Wurtele and Nikolau, Arch. Biochem. Biophys. 278:179, 1990). Neither peak I nor peak II contained biotinylated proteins < 200kDa or utilized methylcrotonyl-CoA as a substrate. However, both enzymes utilized propionyl-CoA approximately 50% as efficiently as acetyl-CoA.

The identity of the 233kDa ACCase polypeptide as the Acc1 gene product was suggested by several observations. First, high concentrations of sethoxydim and haloxyfop inhibit more than 80% of ACCase activity in crude leaf extracts from wildtype but not herbicide-tolerant maize (PNAS 87:7175). Second, immunoprecipitation with antiserum to peak II ACCase decreased ACCase activity by 75% and preferentially removed the 233kDa polypeptide from the crude extract solution. Conclusive evidence that the 233kDa subunit is encoded by the Acc1 gene was obtained by measuring the haloxyfop I50 values for peak I and peak II from both B73 (haloxyfop-sensitive) and S2 (haloxyfop-tolerant) maize leaves. Peak I ACCase (225kDa) was highly tolerant (I50 > 100uM) to haloxyfop in both genotypes and therefore was not correlated with whole plant sensitivity to the herbicide. In contrast, haloxyfop I50 values for peak II ACCase (233kDa) were greater in S2 (11uM) than in B73 (< 1uM), consistent with differences in ACCase inhibition previously reported for wildtype and mutant crude leaf extracts (PNAS 87:7175).

Further efforts will be directed towards sequencing and characterizing putative clones of maize ACCase obtained by antibody screening of a lambda gt11 cDNA library from A188 seedlings. Early evidence indicates that the sequence of a partial cDNA clone for maize ACCase is similar to and co-linear with animal ACCases.


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