Evidence for Energy-Dependent '4C-Photoassimilate Retention in Isolated Tobacco

"C-photoassimilate release from isolated tobacco (Nicotiana tabacum L. cv Xanthi nc.) mesophyll cells was gradual and much of the photoassimilate was retained for several hours. "C-product retention was enhanced by Ca2' and impaired by EDTA and ATP. Mg2' reduced the ATP-enhanced "C-efflux suggesting that ATP impairs product retention by modifying cell membrane permeability, possibly as a result of its chelating properties. Various metabolic inhibitors increased "4C-efflux indicating an energy requirement for "C-product retention. Profiles of"4C-product distribution in the cells and suspending medium indicated that "C-efflux from cells treated with carbonylcyanide p-trifluoromethoxyphenylhydrazone occurred by passive diffusion. Coupled with the observation that exogenous sucrose increased IC-efflux and the demonstration of C-sucrose uptake, the results suggest that product retention is partially due to the activity of an energy-dependent uptake process which recovers much of the photoassimilate lost from the cells. Under certain conditions, carbonylcyanide p-trifluoromethoxyphenylhydrazone reduced "4C-efflux. The apparently contradictory effects of the uncoupler appeared to be related to the intracellular photoassimilate concentration and may be explained by the partitioning of photoassimilate within the cells. A comparison ofthe fluxes for passive permeation and phloem loading in sugar beet has led to discussions concerning the possible involvement of adaptations to increase the flux rates across the membranes ofassimilating cells (8). Energy-dependent secretory processes have been observed in photosynthesizing carrot callus cultures (13) and isolated mesophyll protoplasts of wheat and tobacco (14) where sugar release is ooupled with K+ efflux. Evidence for an energy-dependent release of photoassimilate has also been obtained with leaf fiagments of sugar beet where low 02 concentrations reduce sugar efflux from the assimilating cells while enhancing the rate of CO2 fixation (5). A similar enhancement of CO2 fixation, accompanied by an increase in the rate of translocation, also has been observed by Servaites and Geiger (25) in sugar beet leaves exposed to low 02 concentrations. Thus, it would appear that low 02 levels increase the rate of CO2 fixation and the rate of translocation while simultaneously reducing the rate ofentry of potential translocate into the apoplast in leaves of sugar beet. Unfortunately, the complexities inherent in leaf morphology and physiology result in ambiguities which inevitably complicate the interpretation of experimental results. Consequently, despite recent progress, our understanding of the mechanisms by which photoassimilate moves from the assimilating cells to the conducting tissues within the leaf is far from complete. This study was initiated in an attempt to define further the nature of the processes involved in product retention and efflux from separated tobacco mesophyll cells. The apoplast is considered by some investigators to provide a major route for the movement of photoassimilate from the assimilating cells to the conducting tissues (9, 20, 21). If so, the assimilating cells could influence the products available for translocation by controlling the type of product released into the apoplast. Evidence for discriminatory product release from assimilating cells has been obtained with leaf fragments of sugar beet (3-6,20). A transient asymmetry also was observed between the product profile of separated cotton mesophyll cells and the medium (17). Apoplastic transport also is thought to involve phloem loading from the apoplast by means of energy-dependent uptake mechanisms (6, 9, 10). Consequently, the assimilating cells also could affect the rate oftranslocation by regulating the rate ofassimilate release into the apoplast. The extent of product release from assimilating cells has been examined using isolated mesophyll cells (18, 23), leaf fragments (3, 4, 20, 21), and intact leaves (8). ' Supported by a research studentship from the Science Research Council, U.K., and performed in partial fulfillment of the requirements for the Ph.D. thesis. 2 Present address: Dpto. Genetica y Fisiologia Vegetal, Centro de Investigaci6n Cientifica de Yucatan, A.C. , Apartado Postal 924, Yucatan, Mexico. MATERIALS AND METHODS Plant Culture. Tobacco plants (Nicotiana tabacum L. cv Xanthi nc.) were grown under greenhouse conditions for 10 weeks followed by 2 weeks at 25°C in an environmental chamber with a 12-h photoperiod provided by a bank of fluorescent lamps giving a light intensity of 86 w m-2 at floor level. Cell Isolation. 'Low-loaded' cells were isolated from fully expanded leaves of plants kept in the dark for 12 to 18 h before leaf excision. 'High-loaded' cells were obtained from leaves excised several hours after the start of the photoperiod. Mesophyll cells were isolated according to the method of Jensen et al. (16), substituting 1 mM EDTA for Ca2l in the maceration medium. Following isolation, the cells were washed thoroughly and resuspended in reaction medium (600 mM sorbitol, 50 mM Hepes [pH 7.0]) containing the salts and growth substances of the wash medium (16). "CO2 Fixation. Cells (4-10 ug Chl/ml), illuminated at saturating light intensity (8,600 lux at flask level), were incubated at 25C. CO2 fixation was initiated by the addition of NaH"CO3 (final concentration 8 mM). At timed intervals, aliquots were acidified and the acid-stable radioactivity determined. "C-Efflux. "4C-Labeled cells were washed and resuspended in fresh reaction medium. Aliquots were shaken gently at 25°C and illuminated as described previously. Vials wrapped in two layers