Cloning of hmg1 and hmg2 cDNAs encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase and their expression and activity in relation to alpha-farnesene synthesis in apple

In plants, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyses the synthesis of mevalonate from 3-hydroxy-3-methylglutaryl coenzyme A. It has been reported to be the rate-limiting enzyme in sesquiterpene and trit erpene biosynthesis and is encoded by a small gene family. The accumulation of alpha -farnesene in the skin tissue of apple fruit during storage appea rs to be predominantly through the classical mevalonate pathway and not thr ough the novel glyceraldehyde-3-phosphate/pyruvate pathway independent of H MGR action. The content of a-farnesene in the skin tissue increased during the first 8 weeks of storage at 0 degreesC in air, and started declining af ter 12 weeks. In contrast, HMGR activity in the total membrane and soluble fractions, was the highest at the time of harvest, but decreased during the first 8 weeks in storage and remained stable thereafter. The potent ethyle ne action inhibitor 1-methylcyclopropene inhibited alpha -farnesene evoluti on and HMGR activity by 97 and 30 %, respectively. As a first step in study ing the molecular mechanism of apple HMGR regulation, we have isolated and cloned a full-length cDNA (hmg1) as well as a fragment (hmg2), using apple skin mRNA and RT-PCR in the presence of degenerate oligonucleotides designe d against conserved regions of plant HMGR genes. Genomic Southern analysis using probes designed for the 3'-end of the two cDNA clones confirmed the p resence of at least two HMGR genes in apple. The cDNA for hmgl has an open reading frame of 1 767 nucleotides. Analysis of the nucleotide sequence rev ealed that the cDNA encodes a polypeptide of 589 residues with a relative m olecular mass of 62.7 kDa. The hydropathy profile of the putative polypepti de indicated the presence of two highly hydrophobic domains near the amino terminus. Northern blot analysis confirmed that both hmg1 and hmg2 transcri pts possessed a size of 2.4 kb. The two genes are differentially expressed during low temperature storage and in response to C2H4, with hmg1] being ex pressed constitutively and hmg2 being relatively more sensitive to developm ental stimuli and ethylene. (C) 2001 Editions scientifiques et medicales El sevier SAS.