THE FATE OF OSMO-ACCUMULATED PROLINE IN LEAF DISCS OF RAPE (BRASSICA-NAPUS L) INCUBATED IN A MEDIUM OF LOW OSMOLARITY

When rape leaf discs were submitted in vitro to an upshock osmotic str ess they accumulated proline. We used leaf discs treated for 16-20 h i n the light with either sucrose 800 mM (-2.3 MPa) or PEG 6000 (400 g/k g H2O) (-1.69 MPa) to study their capacity to mobilize proline once tr ansferred to media of higher osmotic potential, It was found that prol ine metabolism took place: with no lag provided the external pressure was increased stepwise by 0.3 MPa. The mean rate of proline metaboliza tion, which was lower than the rate of accumulation during the upshock , was dependent on the level of proline available at the beginning of the transfer and related to the external osmotic potential. In the rec overy medium, following a hyperosmotic stress with sucrose, a signific ant quantity of proline leakage occurred, indicating organic solute ef flux during osmotic adjustment of leaf discs experiencing hypo-osmotic stress. These released solutes were taken up by the leaf disc and mob ilized during the later stages of recovery. Fluxes of carbohydrates an d proline are much less prominent in PEG-treated discs. The kinetics p erformed with sucrose and PEG-treated leaf discs showed that a signifi cant fraction of the osmo-accumulated proline was not available for mo bilization during recovery. It is suggested that, at the cellular leve l, this proline could be stored in the vacuole. This contrasts with th e main fraction, which presumably accumulated in the cytosol/plastids. Such compartmentation seems to be related to the upshock osmotic stre ss response, because turgid leaf discs loaded with exogenous L-proline exhibited a high rate of proline mobilization when transferred to the reference medium not supplemented with proline. As demonstrated by th e changes, firstly, in the level of individual free amino acids during the recovery of the leaf discs, and secondly induced by added citrate and glutamate on the rate of proline withdrawal, proline metabolizati on is partly reliant on conversion to, and the subsequent metabolism o f glutamate. However changes in the activities at the level of transcr iption and protein synthesis are also involved, as shown by the additi on of various inhibitors of protein synthesis or proline analogs.