Aberrant processing of polyphenol oxidase in a variegated grapevine mutant

Area of Science:

• Plant Biochemistry
• Molecular Biology
• Viticulture

Background:

• Grapevine (Vitis vinifera L.) fruit browning is influenced by polyphenol oxidase (PPO) activity.
• Bruce's Sport is a unique mutant variety exhibiting reduced browning in white-variegated fruit tissues.

Purpose of the Study:

• To investigate the molecular basis for reduced PPO activity in Bruce's Sport grapevine.
• To characterize the PPO enzyme and its processing in both wild-type Sultana and mutant Bruce's Sport grapes.

Main Methods:

• Purification and molecular weight determination of active PPO from Sultana grapes using SDS-PAGE.
• Western blot analysis to compare PPO protein levels in Sultana and Bruce's Sport.
• Protease digestion assays to assess PPO precursor processing.
• Northern blot analysis using a grape PPO cDNA probe to examine mRNA levels.

Main Results:

• Active PPO in Sultana grapes is a 40-kilodalton protein, with a minor 60-kilodalton band present in young berries.
• Bruce's Sport grapes exhibit significantly lower levels of the 40-kDa PPO and higher levels of the 60-kDa protein.
• Protease treatment of Bruce's Sport extracts converted the 60-kDa band to the 40-kDa form.
• Both grapevine varieties possess a 2.2-kilobase PPO mRNA transcript, with levels decreasing during berry development.

Conclusions:

• The 40-kDa active PPO in grapevine is synthesized from a precursor protein of at least 60 kDa.
• Bruce's Sport mutation disrupts the normal processing of the PPO precursor, leading to an accumulation of the inactive 60-kDa form and reduced browning capacity.