A putative citrus vacuolar processing thiolprotease cDNA (Citvac) was isolated from a cDNA library of Citrus fruits (Citrus sinensis 1. Osbeck var Washington navel). l h e cDNA is 58 and 57% identical with vacuolar processing seed proteases from castor bean and soybean, respectively. l h e 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 microsoma1 fraction and to undergo proteolytic processing and glycosylation. Transcript levels for the Citvac are developmentally regu- The orange is a nonclimateric fruit, i.e. a sharp increase in respiration and ethylene production does not accompany the ripening process (Rasmussen, 1975). However, the peel of the orange is able to produce significant amounts of ethylene under certain conditions (Riov et al., 1969; Biggs, 1983, 1988). Furthermore, ethylene seems to be required for degreening of this part of the fruit (flavedo), which normally occurs during fruit ripening (Goldschmidt et al., 1993). These processes are associated with alterations at the biochemical and gene expression leve1 (Alonso et al., 1992;Burns and Baldwin, 1994). Treatment of full-size green fruits with ethylene induces a number of changes at the morphological (Shimokawa et al., 1978), physiological, and molecular (Stewart and Wheaton, 1972;Alonso et al., 1992) levels similar to those observed during natural degreening of the fruit that occurs during ripening.In this paper we report the molecular characterization of an mRNA (Citvac) that accumulates in the flavedo tissue of the fruit during ripening. This mRNA encodes a protein homologous to Cys proteinases from castor bean and soy- bean seeds that are involved in posttranslational maturation of vacuolar storage proteins Shimada et al., 1994). The pattern of expression of this mRNA, its regulation by ethylene, and its possible role during fruit ripening will be discussed.
MATERlALS A N D METHODS
Plant Materials and Ethylene TreatmentFruits ( correspond to green fruits, zero values to yellow fruits, and negative values to orange and red fruits. Immature fruits (1 cm in diameter) and flowers at different developmental stages were obtained from the same trees. Samples were harvested, frozen immediately in liquid N,, and stored at -80°C until use. For roots, plants were grown hydroponically in Hoagland solution in the greenhouse. Roots of 1 or 2 mm in diameter were rinsed with water, blotted with filter paper, and frozen immediately in liquid N,. Leaf explants consisted of terminal segments of vegetative branches (about 15 cm) bearing five to eight mature leaves. Explants were quickly transported to the laboratory, and the lower part of the stem was placed in a via1 with water during ethylene treatment. Ethylene treatments were carried out in the dark as described previously (Alonso et al., 1992).
lsolation of Tar4N1A flavedo-enriched fruit cDNA library (E12 library) (Alonso et al., ...