Developmental and light-regulated post-translational control of 3-hydroxy-3-methylglutaryl-CoA reductase levels in potato

Abstract

In plants, the first committed step in the cytosolic pathway for biosynthesis of isoprenoids is catalysed by 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). We have added an eight amino-acid-residue epitope tag to a potato (Solanum tuberosum L.) HMGR isoform and expressed this novel protein (HMGR-FLAG) in transgenic plants. Despite high levels of transcript accumulation in all leaf stages of transgenic plants, high levels of HMGR-FLAG protein were found only in apical meristematic tissue, suggesting post-translational regulation of potato HMGR affected by plant development. Protein immunoblots, and determination of enzymatic activity and transcript accumulation in the HMGR-FLAG transgenic and the non-transgenic parental plant lines, show that HMGR levels decrease dramatically in the dark. Again, the mechanism of this control occurs at a post-translational level. After 2.5 h in darkness, levels of HMGR-FLAG are approximately one-half of those in plants in the light; protein levels recover rapidly when dark-treated plants are returned to the light. In non-transgenic plants, hmg transcript levels are reduced in the dark, whereas dark treatments do not affect transgene hmg transcripts expressed under the control of a constitutive promoter. Furthermore, transcripts for HMGR-FLAG remain associated with polyribosomes in dark-treated tissues. Addition of inhibitors of cysteine proteases during microsomal protein extraction is required for recovery of immunoreactive HMGR-FLAG. The epitope-tagged isozyme has been used to show for the first time that a regulated decrease in plant HMGR activity correlates closely with a loss of the HMGR protein. We have used whole plants to demonstrate that developmental and light-regulated control of HMGR occurs post-translationally in vivo.