An assessment of the physiological properties of the so-called compatible solutes using in vitro experiments with leaf discs

Despite the considerable efforts directed over the past four decades to investigate, via correlative physiology and genetic engineering, the contribution of osmolytes to osmotolerance of higher plants, their physiological function(s) still remain a matter of debate. Here, we summarize the functions of proline and glycine betaine, recently documented in our group by using canola (Brassica napus L. var. oleifera), leaf discs treated in vitro with medium of low or high osmolarities. These discs accumulate proline when subjected to osmotic upshifts, whereas proline is rapidly consumed when the discs are further treated under non-stressing conditions. The period of time needed to osmoregulate the amount of proline is assumed to be too long to assign to the proline response a relevant function in turgor regulation which can be achieved with carbohydrates. In addition, proline accumulation is suppressed by supplying the osmotically stressing medium with polyamines, phyto-oxylipins and glycine betaine with no detrimental effect on the capacity to cope with osmotic stress. In opposition to proline, glycine betaine does not occur in canola. We take advantage of this situation to test the compatibility of exogenously supplied betaine in this plant material. Comparisons are done with spinach (Spinacia oleracea L.) leaf discs, which contain an endogenous amount of betaine and also exhibit the capacity to absorb a large amount of it. The betaine absorbed is not degraded and thus cannot be considered in this situation as a true osmoregulator. In turgid tissues of plants that do not produce betaine, this compound induces an elevated level of glutamine, a decreased level of protein, and photo-dependent glycine accumulation. This can rely on senescent-like effects and on a blockage in the glycolate cycle. Such damages to nitrogen metabolism are not observed in leaf explants treated simultaneously with betaine and the stressing agent. We also find that the treatments of turgid canola leaf discs with proline or betaine induce important changes in polyamine contents, mimicking, but with a higher order of magnitude, the effects of osmotic stress. Interestingly, this inducing activity is not observed when osmolytes are supplied under osmotic upshift conditions. Antagonistic effects between osmolytes and osmotic stress are described for the first time in higher plants. They suggest that proline and betaine can act as signal molecules in counteracting destabilizing effects to polyamine metabolism and lead us to decipher other counter-stress effects of nitrogenous osmolytes which, obviously, do not behave just as inert osmobalancers.