Differential Accumulation of Biotin Enzymes during Carrot Somatic Embryogenesis

Abstract

The activities of four biotin enzymes, acetyl-coenzyme A (CoA) carboxylase, 3-methylcrotonyl-CoA carboxylase, pyruvate carboxylase, and propionyl-CoA carboxylase, and the accumulation of six biotin-containing polypeptides were determined during development of somatic embryos of carrot (Daucus carota). Acetyl-CoA carboxylase activity increased more than sevenfold, whereas the activities of 3-methylcrotonyl-CoA carboxylase, pyruvate carboxylase, and propionyl-CoA carboxylase were relatively unaltered. An increase also occurred in the accumulation of three of the biotin-containing polypeptides (molecular masses of 220, 62, and 34 kilodaltons). Of these, the most dramatic change was in the accumulation of the 62-kilodalton biotin-containing polypeptide, which increased by at least 50-fold as embryogenic cell clusters developed into torpedo embryos. (9). The biotin enzymes propionyl-CoA carboxylase, 3-methylcrotonyl-CoA carboxylase, and pyruvate carboxylase catalyze the ATP-dependent formation of methylmalonyl-CoA, methylglutaconyl-CoA, and oxaloacetate, respectively. These enzymes have only recently been detected in plants (23), and nothing is yet known about their structure, regulation, or metabolic functions.To begin the characterization of each of these enzymes, we investigated the changes in the activities of the biotin enzymes and the accumulation of biotin-containing polypeptides during development. The somatic embryogenesis system of carrot (8) enabled the isolation of large quantities of embryos at specific stages of development (6,7,10,22). We report the changes in the activities of the four biotin enzymes of plants (acetyl-CoA carboxylase, propionyl-CoA carboxylase, 3-methylcrotonyl-CoA carboxylase, and pyruvate carboxylase) and the changes in the accumulation of the biotincontaining polypeptides during carrot somatic embryo development. MATERIALS AND METHODS Cell CulturesCell cultures were initiated from the secondary phloem of carrot roots (Daucus carota var Danver) and maintained essentially as previously described (10,22) Fractionation ProceduresThe (+)2,4-D cultures were separated into embryogenic cell clusters and nonembryogenic cell fractions by Ficoll density gradient centrifugation (22). Embryos at specific developmental

Characterization of Maize Acetyl-Coenzyme A Carboxylase

“…The existente of ACCase isoforms in plants is further suggested by reports of differential sensitivity to aryloxyphenoxypropionate herbicides of [14C]acetate incorporation into short versus long-chain FAs (Walker et al, 1988;Howard and Ridley, 1990), anion-exchange chromatographic separation of two ACCase activities from a hybrid maize variety (Howard and Ridley, 1990), and occurrence of multiple isoforms in rat (Bianchi et al, 1990;Kong et al, 1990). In a recent report, Wurtele and Nikolau (1992) showed that a 7-fold increase in ACCase activity during carrot somatic embryogenesis is associated with a large increase in biotin proteins of 62, 210, and 34 kD, which the authors suggested might represent different ACCase isoform subunits. The 62-kD polypeptide may be related to a 50-kD biotin protein in developing carrot embryos that was described in preliminary reports as an ACCase isoform subunit (Nikolau and Wurtele, 1991) and was used to obtain a cDNA clone that hybridized to a small gene family .…”

“…For example, UV-irradiation of parsley cells induces increased ACCase activity and flavonoid synthesis, but only one ACCase polypeptide is found even though these cells are also likely to synthesize FAs (Egin-Buhler et al, -1980). However, ACCase activities with different properties (Walker et al, 1988;Howard and Ridley, 1990) and multiple biotinylated proteins associated with increased ACCase activity (Wurtele and Nikolau, 1992) have also been described.…”

Targeting of the Arabidopsis Homomeric Acetyl-Coenzyme A Carboxylase to Plastids of Rapeseeds

“…The only known plant biotinylated polypeptides with a molecular mass greater than 200 kD are the HO-ACCase isozymes (Wurtele and Nikolau 1990;Wurtele et al, 1992;Baldet et al, 1993;Duval et al, 1994). Therefore, anti-biotin immunoblots were used to assess the presence or absence of the ACC1 gene product in mature rapeseed (Fig.…”

Molecular Characterization of the Non-biotin-containing Subunit of 3-Methylcrotonyl-CoA Carboxylase

“…We interpret elevated MCCase mRNA levels to reflect higher rates of leucine (1,5,(41)(42)(43) and/or isoprenoid (2,42,44) catabolism. We hypothesize that such increased catabolism is needed to satiate demands for ATP generation, particularly in organs and tissues that are not net photosynthetic.…”