The fate of osmo-accumulated proline in leaf discs of Rape ( Brassica napus L.) incubated in a medium of low osmolarity

Abstract

When rape leaf discs were submitted in vitro to an upshock osmotic stress they accumulated proline. We used leaf discs treated for 16–20 h in the light with either sucrose 800 mM (−2.3 MPa) or PEG 6000 (400 g/kg H 2O) (−1.69 MPa) to study their capacity to mobilize proline once transferred to media of higher osmotic potential. It was found that proline metabolism took place with no lag provided the external pressure was increased stepwise by 0.3 MPa. The mean rate of proline metabolization, 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 recovery medium, following a hypersomotic stress with sucrose, a significant quantity of proline leakage occurred, indicating organic solute efflux during osmotic adjustment of leaf discs experiencing hypo-osmotic stress. These released solutes were taken up by the leaf disc and mobilized during the later stages of recovery. Fluxes of carbohydrates and proline are much less prominent in PEG-treated discs. The kinetics performed with sucrose and PEG-treated leaf discs showed that a significant fraction of the osmo-accumulated proline was not available for mobilization during recovery. It is suggested that, at the cellular level, this proline could be stored in the vacuole. This contrasts with the main fraction, which presumably accumulated in the cytosol/plastids. Such compartmentation seems to be related to the upshock osmotic stress 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 the 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 metabolization is partly reliant on conversion to, and the subsequent metabolism of glutamate. However changes in the activities at the level of transcription and protein synthesis are also involved, as shown by the addition of various inhibitors of protein synthesis or proline analogs.