Isolation and Characterization of cDNA Clones for the E1β and E2 Subunits of the Branched-chain α-Ketoacid Dehydrogenase Complex in Arabidopsis

Abstract

Branched-chain alpha-ketoacid dehydrogenase (BCKDH) has been known in mammals to be a key enzyme of the catabolic pathway of branched-chain amino acids. We have isolated two cDNA clones encoding the E1beta and E2 subunits of BCKDH, respectively, from Arabidopsis thaliana. Proteins encoded in these cDNA sequences had putative mitochondrial targeting sequences and conserved domains reported for their mammalian counterparts. Northern blot and immunoblot analyses showed that transcripts from the respective genes and E2 protein markedly accumulated in leaves kept in the dark. We found that the activity of BCKDH in the leaf extracts also increased when plants were placed in the dark. Addition of sucrose to detached leaves inhibited the accumulation of transcripts, whereas application of a photosynthesis inhibitor strongly induced the expression of these genes even under light illumination. These observations indicate that the cellular sugar level is likely responsible for the dark-induced expression of these genes. The transcript levels of these genes were also high in senescing leaves, in which photosynthetic activity is low and free amino acids from degraded protein are likely to serve as an alternative energy source.