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United States Department of Agriculture

Agricultural Research Service

Title: Cloning and Bacterial Expression of a 3-Hydroxy-3-Methylglutaryl-Coa Reductase Cdna (Hmg1) from Peel Tissue of Apple Fruit

Authors
item Pechous, Steven
item Whitaker, Bruce

Submitted to: Journal of Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 3, 2002
Publication Date: N/A

Interpretive Summary: Superficial scald is a costly cold storage disorder of apples that affects fruit of many popular varieties such as Red Delicious, Granny Smith and McIntosh. Scald is thought to be induced by oxidation products of a volatile compound, alpha-farnesene, which is produced in the peel tissue of apples during storage. To control scald, apples are routinely drenched with a concentrated solution of an antioxidant chemical after harvest. This is expensive and results in unwanted chemical waste and residue on the fruit. Our research is aimed at understanding, at the genetic and biochemical level, why some apples are highly susceptible and others are resistant to the scald disorder. In this study, two genes from apple peel were isolated that code for an enzyme, HMGR, that performs the first step in farnesene synthesis. It was found that activation of HMGR genes is very unlikely to account for the acute rise in farnesene production during the initial weeks of apple storage. The ultimate goal of this work is to limi farnesene synthesis in apples, and thereby prevent scald development, by molecular genetic means. This outcome will benefit both the apple industry and consumers by creating new scald-resistant lines that do not require chemical treatment prior to storage.

Technical Abstract: As part of an effort to elucidate the mechanism of superficial scald in apple fruit, and ultimately to devise a molecular genetic strategy for control of this storage disorder, we have begun to clone and express genes that regulate production of alpha-farnesene in peel tissue. Oxidation products of this sesquiterpene are thought to induce necrosis of cell layers just beneath the fruit skin, leading to development of scald symptoms. Here we report the cloning and bacterial expression of a cDNA encoding 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), the initial, rate-limiting enzyme in sesquiterpene biosynthesis via the mevalonic acid pathway. RT-PCR cloning based on the conserved catalytic domains of plant HMGRs yielded two isoforms from peel tissue mRNA, designated HMG1 and HMG2. The 3' untranslated regions of the two genes, obtained using 3' RACE, were used to screen an apple peel cDNA library. Full-length transcripts of HMG1 1were obtained, whereas only partial HMG2 clones were isolated. RNA-gel blots of HMG1 showed a high level of expression in peel tissue at harvest and after 4 and 8 weeks of storage at 0.5 C, even when tissue responsiveness to ethylene was blocked by prestorage treatment of fruit with 1-methylcyclopropene. Hence, the rapid rise in farnesene production that occurs in the initial weeks of storage cannot be attributed to increased expression of HMG1. E. coli cells transformed with the HMG1 transcript showed over 10-fold higher HMGR activity than cells transformed with the empty expression vector. Bacterial expression was confirmed by immunoblots of HMG1 protein fused to a C-terminal myc tag.

Last Modified: 10/20/2014
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