A Putative Vacuolar Processing Protease Is Regulated by Ethylene and also during Fruit Ripening in Citrus Fruit

A putative citrus vacuolar processing thiolprotease cDNA (Cit-vac) was isolated from a cDNA library of Citrus fruits (Citrus sinensis L. Osbeck var Washington navel). The cDNA is 58 and 57% identical with vacuolar processing seed proteases from castor bean and soybean, respectively. The Citvac sequence shows a typical signal peptide for entering into the endoplasmic reticulum and two glycosylation signals. Using an in vitro transcription-translation system, we show that the Citvac precursor is able to enter a microsomal fraction and to undergo proteolytic processing and glycosylation. Transcript levels for the Citvac are developmentally regulated in the flavedo (outer colored part of the fruit peel) and increase during fruit ripening and in the flower during opening. Exogenous treatment with ethylene induces Citvac mRNA expression in both fruits and leaves. Citvac is encoded by one or two genes in the Citrus genome. The possible role of the Citvac gene product during fruit ripening and other ethylene-mediated processes is discussed.