420 J. AMER. SOC. HORT. SCI. 125(4):420–424. 2000. J. AMER. SOC. HORT. SCI. 125(4):420–424. 2000. PP i Formation by Reversal of the Tonoplast-bound H + -pyrophosphatase from ‘Valencia’ Orange Juice Cells Kenneth Marsh, 1 Pedro Gonzalez, and Ed Echeverría 2 University of Florida, Institute of Food and Agricultural Sciences, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850 ADDITIONAL INDEX WORDS. vacuole, fruit development, Citrus sinensis ABSTRACT. Tonoplast vesicles isolated from juice cells of mature ‘Valencia’ oranges [Citrus sinensis (L.) Osbeck] showed similar tonoplast-bound vacuolar ATPase (V-ATPase) and inorganic pyrophosphatase (V-PPiase) activity as measured by product formation. Both proton pumps were able to generate a similar pH gradient, although steady-state was reached faster with ATP as substrate. When a ∆ pH of 3 units was imposed (vesicle lumen pH of 4.5 and incubation medium of 7.5), tonoplast-bound PPiase was not able to significantly amplify the existing ∆pH. Although not able to function as a H + pump, V-PPiase effectively synthesized PPi in the presence of inorganic phosphate (Pi). Formation of PPi by V-PPiase was enhanced by ATP but inhibited by NaF, gramicidin, and by antibodies raised against V-PPiase from mung bean [Vigna radiata (L.) R. Wilcz. (Syn. Phaseolus aureus Roxb.)]. Immunological analysis demonstrated an increase in V-PPiase protein with fruit maturity. Data indicate that under in vivo conditions, the V-PPiase of mature orange juice cells acts as a source of inorganic pyrophosphate (PPi) but not as a H + pump. We propose that synthesis of PPi provides a mechanism for recovery of stored energy in the form of the pH gradient across the vacuole during later stages of development and postharvest storage. glycolytic synthesis of fructose-1,6-P 2 (Mertens et al., 1990) thus becoming a mechanism for ATP conservation. Under similar anaerobic conditions, sucrose breakdown occurs seemingly through the sucrose synthase (SS) pathway (Perata et al., 1996; Ricard et al., 1991). Both the activity of PFP and conversion of UDPG (product of sucrose synthase) to glucose-1-P require a steady supply of PPi inasmuch as its cytosolic levels remain unchanged even during marked respiratory fluxes (Dancer and ap Rees, 1989; Weiner et al., 1987). It was observed recently that tonoplast-bound PPiase is over-expressed in response to ener- getic stress such as anoxia and chilling in rice (Oryza sativa L.) seedlings (Carystinos et al., 1995) and mung bean [Vigna radiata (syn. Phaseolus aureus)] hypocotyl (Darley et al., 1995). A role for V-PPiase in the supply of PPi was demonstrated for maize (Zea mays L.) seeds and coleoptiles under similar conditions (Rocha and de Meis, 1998). These observations suggest that V- PPiase may play a role in the supply of PPi under anaerobic conditions (limited ATP) occurring during later stages of citrus fruit maturity. Preliminary studies have established the presence of PPiase in tonoplast vesicles isolated from sweet orange juice cells. Given the fact that V-PPiase can not act in the hydrolytic direction under conditions found in mature citrus fruit, we exam- ined the possibility of this tonoplast-bound H + pump acting in the direction of PPi formation during vacuole deacidification, there- fore becoming a PPi source for PFP and glucose-1-P production. In this report, we present evidence demonstrating the synthesis of PPi coupled to the efflux of protons by ‘Valencia’ orange juice cell tonoplast V-PPiase. Materials and Methods PLANT MATERIAL. Mature ‘Valencia’ oranges were collected in early April 1999 from groves located at the Citrus Research and Education Center, Lake Alfred, Fla. Fruit were transported to the laboratory and used immediately for tonoplast extraction. TONOPLAST VESICLE EXTRACTION. Tonoplast vesicles were iso- lated in a discontinuous sucrose gradient following the procedure described previously for sweet limes (Citrus limmetioides Tanaka) (Echeverria et al., 1997). After isolation, tonoplast vesicles were Received for publication 25 Oct. 1999. Accepted for publication 7 Apr. 2000. Florida Agricultural Experiment Station journal series R-07192. We are grateful to M. Maeshima for the gift of antibodies used in this study. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must be hereby marked advertisement solely to indicate this fact. 1 Current address: Horticulture and Food Research Institute of New Zealand, Private Bag 92-169, Auckland, New Zealand. 2 To whom reprint requests should be addressed; eje@lal.ufl.edu. The early stages in the development of citrus fruit (Citrus L. sp.) are characterized by a massive accumulation of citric acid and a parallel decline in vacuolar pH. In sweet oranges (Citrus sinensis), for example, the concentration of citric acid reaches levels of up to 115 mM (Clements, 1964) with vacuolar pH dropping to 2.8 or lower (Echeverria and Burns, 1989). A pH gradient (∆pH) between the acidic vacuole and the neutral cytosol of such magnitude can only be generated by the V-ATPase (Davies, 1994; Schmidt and Briskin, 1993) despite the existence of two H + pumps at the tonoplast of plant cells (i.e., V-ATPase and V-PPiase; Rea and Sanders, 1987). Thermodynamic constraints prevent the V-PPiase from operating in the hydrolytic mode under these extreme physiological condi- tions (Schmidt and Briskin, 1993). Later in citrus fruit development, and continuing throughout postharvest storage, vacuolar citric acid content declines with a concomitant increase in pH (Clements, 1964; Harding and Lewis, 1941; Ting and Vines, 1966; Yamaki, 1990). As the fruit matures, some anatomical and physical characteristics of the pericarp de- velop into effective gas barriers resulting in partial oxygen depriva- tion and decreased aerobic respiration in the interior juice cells (Bain, 1958; Hirai and Ueno, 1977). This increase in anaerobic respiration is evidenced by rising levels of ethanol and acetaldehyde in maturing (Davis, 1970; Roe et al., 1984) and stored fruit (Davis, 1970; Davis et al., 1973). In plant cells, in response to low oxygen pressure, cytosolic ATP content declines with a concomitant in- crease in the ADP/ATP ratio. A marked decline in the energy status (ATP content) of the juice cells has long been recognized to occur in mature citrus fruit (Bruemmer and Roe, 1985). In plants with low ATP levels, PPi dependent phosphofruc- tokinase (EC 2.7.1.90; PFP) acquires a dominant role in the