Characterization of Arabidopsis Fluoroacetate-resistant Mutants Reveals the Principal Mechanism of Acetate Activation for Entry into the Glyoxylate Cycle

The toxic acetate analogue monofluoroacetic acid was employed to isolate Arabidopsis tDNA-tagged plants deficient in their ability to utilize or sense acetate. Several tDNA-tagged lines were isolated, including two that were determined to be allelic to an EMS-mutagenized line denoted acn1 for ac non-utilizing. Following conventions, the tDNA-tagged mutants were designated acn1-2 and acn1-3. Both mutants displayed identical behavior to acn1-1 on a variety of fluorinated and nonfluorinated organic acids, indicating that resistance was specific to fluoroacetate. Thermal asymmetric interlaced PCR identified the sites of tDNA insertion in both mutants to be within different exons in a gene, which encoded a protein containing an AMP-binding motif. Reverse transcription-PCR confirmed that the gene was not expressed in the mutants, and quantitative reverse transcription-PCR showed that the gene is expressed in imbibed seeds and increases in amount during establishment. The wild type AMP-binding protein cDNA was cloned and expressed in Escherichia coli, and the expressed protein was purified by nickel chelate chromatography. The enzyme was identified as an acyl-CoA synthetase that was more active with acetate than butyrate and was not active with fatty acids longer than C-4. The enzyme was localized to peroxisomes by enzymatic analysis of organellar fractions isolated by sucrose density gradient centrifugation. Labeling studies with [14C]acetate showed that acn1 seedlings, like those of the isocitrate lyase mutant icl-1 (isocitrate lyase), are compromised in carbohydrate synthesis, indicating that this enzyme is responsible for activating exogenous acetate to the coenzyme A form for entry into the glyoxylate cycle. The toxic acetate analogue monofluoroacetic acid was employed to isolate Arabidopsis tDNA-tagged plants deficient in their ability to utilize or sense acetate. Several tDNA-tagged lines were isolated, including two that were determined to be allelic to an EMS-mutagenized line denoted acn1 for ac non-utilizing. Following conventions, the tDNA-tagged mutants were designated acn1-2 and acn1-3. Both mutants displayed identical behavior to acn1-1 on a variety of fluorinated and nonfluorinated organic acids, indicating that resistance was specific to fluoroacetate. Thermal asymmetric interlaced PCR identified the sites of tDNA insertion in both mutants to be within different exons in a gene, which encoded a protein containing an AMP-binding motif. Reverse transcription-PCR confirmed that the gene was not expressed in the mutants, and quantitative reverse transcription-PCR showed that the gene is expressed in imbibed seeds and increases in amount during establishment. The wild type AMP-binding protein cDNA was cloned and expressed in Escherichia coli, and the expressed protein was purified by nickel chelate chromatography. The enzyme was identified as an acyl-CoA synthetase that was more active with acetate than butyrate and was not active with fatty acids longer than C-4. The enzyme was localized to peroxisomes by enzymatic analysis of organellar fractions isolated by sucrose density gradient centrifugation. Labeling studies with [14C]acetate showed that acn1 seedlings, like those of the isocitrate lyase mutant icl-1 (isocitrate lyase), are compromised in carbohydrate synthesis, indicating that this enzyme is responsible for activating exogenous acetate to the coenzyme A form for entry into the glyoxylate cycle. During seed germination and establishment, catabolism of fatty acids by glyoxysomal β-oxidation produces large quantities of acetate as acetyl-CoA, which is converted to sucrose via the sequential actions of the glyoxylate cycle, trichloroacetic acid cycle, and gluconeogenesis (1Beevers H. Stumpf P.K. The Biochemistry of Plants. 4. Academic Press, Inc. New York1980: 117-130Google Scholar, 2Gerhard B. Moore T.M. Lipid Metabolism in Plants. CRC Press, Inc., Boca Raton, FL1993: 527-565Google Scholar). In seeds, in which oil is stored in the endosperm, storage lipid is converted completely to sucrose (3Canvin D.T. Beevers H. J. Biol. Chem. 1961; 236: 988-995Abstract Full Text PDF PubMed Google Scholar). Recent reports (4Eastmond P.J. Graham I.A. Trends Plant Sci. 2001; 6: 72-77Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar) suggest that acetate utilization during seedling establishment of some oilseed species may be more complex than described by classical gluconeogenic models. In Arabidopsis, exogenous acetate is respired within mitochondria as well as being converted to sucrose (5Eastmond P.J. Germain V. Lange P.R. Bryce J.H. Smith S.M. Graham I.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5669-5674Crossref PubMed Scopus (225) Google Scholar). An Arabidopsis mutant lacking a mitochondrial carnitine acyl carrier (CAC) 1The abbreviations used are CAC, carnitine acyl carrier; AcetCS, acetyl-CoA synthetase; FAc, monofluoroacetic acid; RT, reverse transcription; Ni-NTA, nickel-nitrilotriacetic acid; MES, 4-morpholineethanesulfonic acid; EMS, ethylmethanesulfonate. protein cannot establish itself, indicating that alternative mechanisms of acyl-CoA or acetyl-CoA metabolism during seedling establishment are required (6Lawand S. Dorne A-J. Long D. Coupland G. Mache R. Carol P. Plant Cell. 2002; 14: 2161-2173Crossref PubMed Scopus (58) Google Scholar). The importance of acetate as a respiratory metabolite has been demonstrated also by acetate/glucose diauxic growth studies of rice cell cultures (7Lee T.K. Lee W.S. Plant Physiol. 1996; 110: 465-470Crossref PubMed Scopus (10) Google Scholar), where acetate was found to be utilized preferentially by inhibiting glucose uptake (8Lee T.K. Lee S.M. Lee W.S. Plant Cell Physiol. 1999; 40: 1046-1052Crossref Scopus (8) Google Scholar). The preferential use of acetate was accompanied by an increase in the activity of glyoxylate cycle enzyme isocitrate lyase, which supported the proposal that acetate was a positive regulator of glyoxylate cycle enzyme and gene expression in plants (9Kudielka R.A. Theimer R.R. Plant Sci. Lett. 1983; 31: 237-244Crossref Scopus (22) Google Scholar, 10Graham I.A. Leaver C.J. Smith S.M. Plant Cell. 1992; 4: 349-357Crossref PubMed Scopus (129) Google Scholar). Acetate has also been shown to inhibit photosynthetic gene expression and may help regulate the switch from heterotrophy to autotrophy in developing seedlings (11Sheen J. Plant Cell. 1990; 2: 1027-1038Crossref PubMed Scopus (565) Google Scholar). We have undertaken a program to identify Arabidopsis mutants disrupted in their ability to sense acetate as a regulatory metabolite affecting gene expression (12Hooks M.A. Turner J.E. Murphy E. Graham I.A. Mol. Gen. Genomics. 2004; 271: 249-256Crossref PubMed Scopus (19) Google Scholar). We have adapted for Arabidopsis a method employing the biotoxin monofluoroacetic acid, which was successfully used to isolate the acetate regulatory gene facB from Emericella (Aspergillus) nidulans (13Apirion D. Genet. Res. 1965; 6: 317-329Crossref PubMed Scopus (66) Google Scholar, 14Todd R.B. Murphy R.L. Martin H.M. Sharp J.A. Davis M.A. Katz M.E. Hynes M.J. Mol. Gen. Genet. 1997; 254: 495-504Crossref PubMed Scopus (60) Google Scholar). In addition to identifying an important acetate regulatory gene, studies of fluoroacetate-resistant mutants of filamentous fungi have revealed many acetate metabolism genes required for growth on acetate as a sole carbon source, such as those encoding glyoxylate cycle enzymes (15Flavell R.B. Fincham J.R.S. J. Bacteriol. 1968; 95: 1056-1062Crossref PubMed Google Scholar, 16Owen N.E. Chaure P.T. Connerton I.F. J. Gen. Microbiol. 1992; 138: 2599-2608Crossref PubMed Scopus (11) Google Scholar). The acetate nonutilizing mutants, in conjunction with fatty acid nonutilizing mutants (17De Lucas J. Valenciano S. Domínguez A.I. Turner G. Laborda F. Arch. Microbiol. 1997; 168: 504-512Crossref PubMed Scopus (22) Google Scholar), have proven to be valuable tools used to develop detailed models of acetate utilization in filamentous fungi (18De Lucas J. Domínguez A.I. Valenciano S. Turner G. Laborda F. Arch. Microbiol. 1999; 171: 386-396Crossref PubMed Scopus (36) Google Scholar). By employing the screening strategy of resistance to FAc, we predicted that we would also identify plant acetate utilization genes as well as potential regulatory genes. Characterization of two tDNA-tagged mutants revealed independent mutations in a short-chain acyl-CoA synthetase gene of unknown biological function (19Shockey J. Fulda M.S. Browse J. Plant Physiol. 2003; 132: 1065-1076Crossref PubMed Scopus (134) Google Scholar). Based on resistance to FAc, we hypothesized that the enzyme may activate exogenously supplied acetate for entry into the glyoxylate cycle in establishing seedlings (12Hooks M.A. Turner J.E. Murphy E. Graham I.A. Mol. Gen. Genomics. 2004; 271: 249-256Crossref PubMed Scopus (19) Google Scholar). We demonstrate that the enzyme is responsible for this process, and we discuss the function of the enzyme relating to potential sources of acetate production within cells. Plant Material and Genetic Screen—Eighty three seed batches from the Weigel, tDNA mutagenized collection (20Weigel D. Ahn J.H. Blázquez M.A. Borevitz J.O. Christensen S.K. Frankhauser C. Ferrándiz C. Kardailsky I. Malancharuvil E.J. Neff M.M. Nguyen J.T. Sato S. Wang R.A. M.J. C.J. J. Plant Physiol. 2000; PubMed Scopus Google Scholar) were from the Arabidopsis seeds were and imbibed in the for seeds were of and F. Physiol. Scopus Google Scholar), and The to the addition of and were to with the seeds from were containing acid was as a and to the seedlings were seedlings were to described in this be in a for and germination of the mutants the were as described in (12Hooks M.A. Turner J.E. Murphy E. Graham I.A. Mol. Gen. Genomics. 2004; 271: 249-256Crossref PubMed Scopus (19) Google Scholar). of for resistance was a of growth studies were by with the of acetate and butyrate were used for of seedling as not seed batches the wild and mutants seedling of seedlings were and the seedling of and Characterization of was isolated from from acn1-2 and the Thermal PCR Plant J. PubMed Scopus Google Scholar) was on a or and were identical to those that a cycle of for and for was the of the and PCR PCR and were used in conjunction with the tDNA were used for the and PCR as and PCR were cloned into to were by analysis J. Res. 1997; PubMed Scopus Google Scholar) Arabidopsis in The Arabidopsis tDNA was determined by of a which the within the and was to the The for for and for were used in conjunction with the or and to the of and was isolated from seedling and the or the method R. A. J. 236: PubMed Scopus Google Scholar). was on the Reverse and to Reverse was in a containing of of and of reverse The was for PCR was in a containing of PCR sense and The were as of and by was to S. Graham S. Graham I.A. Plant Cell. 2004; PubMed Scopus Google Scholar). The for reverse of of and of reverse was to a of The was for in to the and was on for The reverse was and the was for PCR was on the PCR The sense and for the were and The sense and for acn1 were and in Escherichia and cDNA into with isolated from G. Cell. Full Text PDF PubMed Scopus Google Scholar) was from the Arabidopsis The cDNA was from the by PCR the and which have and The was cloned into the and sites of the expression and the was into E. cells. was expressed and on to and of seedling was as described by (5Eastmond P.J. Germain V. Lange P.R. Bryce J.H. Smith S.M. Graham I.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5669-5674Crossref PubMed Scopus (225) Google Scholar) with the The used of seedlings, which were in a with in of of were an to sucrose density gradient on a of The gradient was for in a an synthetase activity was in a with and to and A. J. Arch. PubMed Scopus (22) Google Scholar), or by short-chain fatty acids to PubMed Scopus Google Scholar). The was to than the of the The was an that required of from by from the and a of the amount of such as enzyme may have also to the was to that enzyme was acyl-CoA activity was to and Res. PubMed Scopus Google Scholar). of and was determined to N.E. 31: PubMed Scopus Google Scholar). protein amount was to M.M. PubMed Scopus Google Scholar) as were determined to and A. 1965; PubMed Scopus Google Scholar). Acetate [14C]acetate were adapted from (5Eastmond P.J. Germain V. Lange P.R. Bryce J.H. Smith S.M. Graham I.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5669-5674Crossref PubMed Scopus (225) Google Scholar) with the Arabidopsis seedlings were for in a containing of and MES, of were used to respired Both fractions were for the seedlings were and (3Canvin D.T. Beevers H. J. Biol. Chem. 1961; 236: 988-995Abstract Full Text PDF PubMed Google Scholar), and the of in was determined on a of The was a Material J. The was in of of and of tDNA of seed of tDNA lines (20Weigel D. Ahn J.H. Blázquez M.A. Borevitz J.O. Christensen S.K. Frankhauser C. Ferrándiz C. Kardailsky I. Malancharuvil E.J. Neff M.M. Nguyen J.T. Sato S. Wang R.A. M.J. C.J. J. Plant Physiol. 2000; PubMed Scopus Google Scholar) two seed lines that were well in the of The seedlings were the of seeds germination seed and and seedlings, were PCR of the of mutant showed that those from were A analysis of of the mutants to the wild type demonstrated that was with to the was of a of function of the mutants to the mutant acn1 demonstrated of the mutations with and being (12Hooks M.A. Turner J.E. Murphy E. Graham I.A. Mol. Gen. Genomics. 2004; 271: 249-256Crossref PubMed Scopus (19) Google Scholar). the mutant was to as acn1-1 and the tDNA-tagged as acn1-2 and The mutants were for the of by growth on a variety of which may be The were to that of used in the for which was used in to of the type of acn1-2 and to the of acetate and butyrate were for both mutants that to was not the of resistance of both mutants to demonstrated that activity or mechanisms of were not compromised in the mutants and were not the of with was wild type and mutants, which that resistance to was a specific of acn1-2 were with those for the wild and and the mutant acn1-1 in the of of the acids acetate and butyrate Both wild and both mutants to the exogenous acetate of and wild type growth was that of the mutants was this acetate in the mutants were those in the wild not that the mutants are more to exogenous acetate than wild type and may be disrupted in an important of acetate utilization and acetate in the mutant would to a of the acid of acetate. Arabidopsis seedlings were also to exogenous butyrate a in seedling with that on acetate was for both wild an exogenous butyrate of a of the on growth was not this and was the to were the two butyrate and were not to be determined in the mutants not A of seedlings to butyrate to acetate would a ability to and Characterization of tDNA for acn1-2 and PCR Plant J. PubMed Scopus Google Scholar) was on acn1-2 and to cDNA containing the of the tDNA and utilized a of three a of and in to a cDNA that the The from the was cloned and to the the J. Res. 1997; PubMed Scopus Google Scholar) analysis of the revealed that for both lines in a AMP-binding protein on the of is the gene by (19Shockey J. Fulda M.S. Browse J. Plant Physiol. 2003; 132: 1065-1076Crossref PubMed Scopus (134) Google Scholar). acn1-2 and from in exons and The acn1-2 a tDNA Both were in the with the the of The tDNA was the of the tDNA been by The a with a and analysis of resistance for of the mutants, to showed that was insertion within not The of the within such that are both demonstrated that resistance was not to expression of a by the which the of analysis of was in to the of in the mutants and to the in developing seedlings of wild type showed that was expressed in seedlings and of wild reports (19Shockey J. Fulda M.S. Browse J. Plant Physiol. 2003; 132: 1065-1076Crossref PubMed Scopus (134) Google Scholar), was not within of the A quantitative was employed to seedling of were with the S. Graham S. Graham I.A. Plant Cell. 2004; PubMed Scopus Google Scholar, Lett. 2003; PubMed Scopus Google Scholar). with the used an expression that that of not The that was expressed in imbibed seeds was with the ability of to seed The expression to and in as shown by the expression in and from and of disrupted gene a predicted protein of acids with a of acids the predicted protein showed to the of and with the and the Turner G. J. 1990; PubMed Scopus Google Scholar). The is required for the of acids E. Mol. Gen. Genet. PubMed Scopus Google Scholar, D. P. 2001; PubMed Scopus Google Scholar). the indicating that is to By an E. (19Shockey J. Fulda M.S. Browse J. Plant Physiol. 2003; 132: 1065-1076Crossref PubMed Scopus (134) Google Scholar) shown that the enzyme a of A synthetase activity with acetate. A analysis of the of that was to be a enzyme and would be to in E. and on was confirmed by PCR of the and in E. as a and showed that and and of the E. protein a than The purified protein showed activity in a with and from the were than of acetate converted to acetyl-CoA in by of The of for short-chain fatty acids was determined by a and short-chain fatty acids The enzyme was active with both acetate and butyrate and was not active with the specific with acetate as were than those determined the of not increase that the are a of of to that the enzyme would be in was confirmed by an analysis of activity in peroxisomes from both wild type and mutant seedlings isolated on sucrose density The sucrose density gradient peroxisomes well from the and mitochondria as shown by an analysis of the enzymes short-chain acyl-CoA and activity was determined in fractions to the of of of the were determined in to of activity in from both wild and mutants and to the different organellar activity was found in the of wild type seedlings Stumpf P.K. Arch. PubMed Scopus Google Scholar), activity was to be in the mitochondrial fractions in wild or In activity was in the of the indicating that this activity was not in the The specific activity of in the wild type seedlings was to that in completely lacking in the which showed that the mutations in the for an independent The specific activity of short-chain acyl-CoA in the was than that in the specific activity of in the was to or than that in short-chain acyl-CoA activity was an of by we that a of activity not for the The activity in the was not of by the to during the was from a of within the the was on seedlings that were to by to for of to was from a of activity and to fractions of density mitochondria and peroxisomes N.E. 31: PubMed Scopus Google Scholar). the would have been from a in fractions containing than sucrose C. J. Plant Physiol. Scopus Google Scholar, 2003; Scopus Google Scholar). a and were not with a the of for isolated by classical gradient Scopus Google Scholar). Acetate by of to peroxisomes that this enzyme is responsible for activating exogenous acetate for entry into the glyoxylate cycle. We this by the metabolism of by acn1-1 with the isocitrate lyase icl-1 (isocitrate (5Eastmond P.J. Germain V. Lange P.R. Bryce J.H. Smith S.M. Graham I.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5669-5674Crossref PubMed Scopus (225) Google Scholar). The acn1-1 mutant was with icl-1 the wild type identical were for the acn1-2 mutant with the isolate not The of in of icl-1 were to those by (5Eastmond P.J. Germain V. Lange P.R. Bryce J.H. Smith S.M. Graham I.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5669-5674Crossref PubMed Scopus (225) Google Scholar) of the in the of the with the of in acids, and organic acids The amount of in the of acn1-1 was by with a increase in the of in the organic acid to acn1-1 and icl-1 were increase was for in and the amount of in the acid was not was with that in the amount of in the lipid was than in or the icl-1 acn1 and icl-1 in the of acetate into the were not the in the icl-1 mutant would have to acetyl-CoA within the In the metabolism of exogenous acetate by icl-1 was than by acn1-1 or wild The for this is not The and those of (5Eastmond P.J. Germain V. Lange P.R. Bryce J.H. Smith S.M. Graham I.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5669-5674Crossref PubMed Scopus (225) Google Scholar) for the icl-1 such as the amount of in and the of in the organic acid and use of employed In both fungi and the of acetate via the glyoxylate cycle to The by (13Apirion D. Genet. Res. 1965; 6: 317-329Crossref PubMed Scopus (66) Google Scholar) isolated the which was deficient in the acetyl-CoA synthetase activity to exogenous acetate into the glyoxylate cycle. The that seed of plants exogenous acetate via the glyoxylate cycle (3Canvin D.T. Beevers H. J. Biol. Chem. 1961; 236: 988-995Abstract Full Text PDF PubMed Google Scholar, C. Stumpf P.K. J. Biol. Chem. Full Text PDF PubMed Google Scholar) that plants an enzyme to activate acetate. The plant enzyme been In a we employed the toxic acetate analogue to for EMS-mutagenized Arabidopsis mutants and we isolated a completely of the (12Hooks M.A. Turner J.E. Murphy E. Graham I.A. Mol. Gen. Genomics. 2004; 271: 249-256Crossref PubMed Scopus (19) Google Scholar). of to of exogenous acn1-1 was the to have the this synthetase a two mutants with identical to acn1-1 and A analysis demonstrated both tDNA mutants were allelic to PCR revealed the sites of insertion to be within the AMP-binding protein which a M.A. A. Graham I.A. Plant Academic Scholar). The of was confirmed in this Characterization of the E. expressed and purified protein demonstrated short-chain acyl-CoA synthetase activity with acetate being a than with the for this enzyme for by (19Shockey J. Fulda M.S. Browse J. Plant Physiol. 2003; 132: 1065-1076Crossref PubMed Scopus (134) Google Scholar), which showed the enzyme more active with butyrate than acetate. The in may be to their use of an E. to we used the is that the acn1 has a more on acetate metabolism which with the The of wild type seedlings to butyrate or mechanisms for acetate may be in PubMed Scopus Google Scholar) as well as in in acetate metabolism Arabidopsis and filamentous fungi are studies with E. (13Apirion D. Genet. Res. 1965; 6: 317-329Crossref PubMed Scopus (66) Google Scholar) that two of acetate with the from the studies suggest mechanisms of acetate within establishing Arabidopsis seedlings The of the glyoxylate to acetate and acetyl-CoA in Arabidopsis been by the that the Arabidopsis mutant icl-1 lipid and respired exogenous acetate (5Eastmond P.J. Germain V. Lange P.R. Bryce J.H. Smith S.M. Graham I.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5669-5674Crossref PubMed Scopus (225) Google Scholar). a the for from to mitochondria for A for this has been that a mitochondrial protein is for seedling establishment in the (6Lawand S. Dorne A-J. Long D. Coupland G. Mache R. Carol P. Plant Cell. 2002; 14: 2161-2173Crossref PubMed Scopus (58) Google Scholar). with the acetate for E. acetyl-CoA mitochondria by a protein (18De Lucas J. Domínguez A.I. Valenciano S. Turner G. Laborda F. Arch. Microbiol. 1999; 171: 386-396Crossref PubMed Scopus (36) Google Scholar). which is in has been isolated by the Arabidopsis mutant may not be and may form of the of acetate and Graham (4Eastmond P.J. Graham I.A. Trends Plant Sci. 2001; 6: 72-77Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar) that the glyoxylate cycle an in Arabidopsis seedling to lipid an has been S.M. Trends Plant Sci. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), of carbon from lipid or acetate to the trichloroacetic acid cycle by of the glyoxylate cycle is and may be a function of the cycle. the function of the glyoxylate cycle is to the of carbon for gluconeogenesis S.M. Trends Plant Sci. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In this a function to as the in the metabolism of exogenous acetate by the glyoxylate cycle. of exogenous acetate in an glyoxylate cycle P.J. Germain V. Lange P.R. Bryce J.H. Smith S.M. Graham I.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5669-5674Crossref PubMed Scopus (225) Google and this we that of that acetate is converted to acetyl-CoA by we have identified a of acetate for entry into the glyoxylate cycle, the of exogenous acetate is not in the is to of acetate the mitochondrial protein with the acetyl-CoA as being by the lyase, which would from Plant Physiol. 2002; PubMed Scopus Google Scholar). this would not to be within the acn1 mutants that glyoxysomal is to that we not activity in glyoxysomal fractions from seedlings of the mutants, glyoxysomal enzyme was identified by a analysis of from I. Plant Cell Physiol. 2003; PubMed Scopus Google Scholar). The activity of this enzyme be we that the may a which a exogenous acetate may be to acetyl-CoA for into the mitochondria by the would the glyoxylate cycle and may exogenous acetate to be respired and in both the acn1 and is to the for E. nidulans by Lucas (18De Lucas J. Domínguez A.I. Valenciano S. Turner G. Laborda F. Arch. Microbiol. 1999; 171: 386-396Crossref PubMed Scopus (36) Google Scholar), acetate is to acetyl-CoA in the to be converted to for into is that the acetate during germination and establishment of Arabidopsis seedlings is more complex than the gluconeogenic the glyoxylate cycle. A as to the of acetate during germination that would the of A potential to acetate has been is during the of and and of J. Plant J. 2000; PubMed Google Scholar). We are the of the acn1 on the of acetate and metabolite by that of acetate are within seedlings, A. D. and A. which may be as as than of acetyl-CoA J. Plant Physiol. 2004; PubMed Scopus Google Scholar). The in the of acetate to acetyl-CoA in seedlings, a that may more important as lipid of acetate may two may be to carbon during the and the may be to acetate as a toxic acid to We the of for with the quantitative We Graham and Smith and for their and Smith for with the We also the Arabidopsis and Arabidopsis for the seed was in with and