Objective:
Analyze the two different 2-kb promoters derived from genomic clones of the apple alpha-farnesene synthase gene AFS1 to determine the mechanism of ethylene- and cold-induced expression in cold-stored apple fruit. Transform tissue from a scald-susceptible apple cultivar with an RNAi construct of AFS1, regenerate transformed plants, and test the transformants for supression of ethylene-and cold-induced alpha-farnesene synthesis.

Approach:
Analysis of the two different AFS1 promoter sequences cloned from leaf tissue genomic DNA of `Law Rome¿ apple will entail testing the expression, in Arabidopsis, tomato, or petunia, of GUS fusion constructs including 0.5- to 2.0-kb fragments of the AFS1 5¿ upstream sequence. Plants transformed with a sense construct of the AFS1 cDNA driven by the AFS1 promoter will be tested for ethylene- and cold-inducible alpha-farnesene synthesis. RNAi gene silencing constructs of AFS1 driven by the CaMV 35S, AFS1, or other suitable plant gene promoter will be used in Agrobacterium-mediated transformation of tissue from a scald-susceptible apple cultivar. Positive transformants screened by DNA gel blot analysis will be propagated and eventually compared with empty vector tranformed controls for ethylene- and cold-inducible alpha-farnesene production in leaf and fruit peel tissues.