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Arabidopsis thaliana cell suspension cultures have been used to investigate the effects of salinity and hyperosmotic stress on plant cellular proteins. We show that 200 mM NaCl and 400 mM sorbitol treatments induce extracellular medium acidification in Arabidopsis cell cultures, a typical response of plant cells to salt and hyperosmotic stress. Using (35)S-labelled amino acids, we demonstrated that NaCl causes a transient suppression of de novo protein synthesis, from which the cells recover within 4 h. Changes in the abundance of cellular proteins 6 h post NaCl and sorbitol treatments were analysed by 2-DE. Of a total of 2,949 protein spots detected on the gels, 266 showed significant changes in abundance across five independent experiments. Using MALDI-TOF MS, we identified 75 salt and sorbitol responsive spots. These fall into 10 functional categories that include H(+) transporting ATPases, signal transduction related proteins, transcription/translation related proteins, detoxifying enzymes, amino acid and purine biosynthesis related proteins, proteolytic enzymes, heat-shock proteins, carbohydrate metabolism-associated proteins and proteins with no known biological functions.

SUMMARYExtracellular ATP regulates higher plant growth and adaptation. The signalling events may be unique to higher plants, as they lack animal purinoceptor homologues. Although it is known that plant cytosolic free Ca 2+ can be elevated by extracellular ATP, the mechanism is unknown. Here, we have studied roots of Arabidopsis thaliana to determine the events that lead to the transcriptional stress response evoked by extracellular ATP. Root cell protoplasts were used to demonstrate that signalling to elevate cytosolic free Ca 2+ is determined by ATP perception at the plasma membrane, and not at the cell wall. Imaging revealed that extracellular ATP causes the production of reactive oxygen species in intact roots, with the plasma membrane NADPH oxidase AtRBOHC being the major contributor. This resulted in the stimulation of plasma membrane Ca 2+-permeable channels (determined using patch-clamp electrophysiology), which contribute to the elevation of cytosolic free Ca 2+ . Disruption of this pathway in the AtrbohC mutant impaired the extracellular ATP-induced increase in reactive oxygen species (ROS), the activation of Ca 2+ channels, and the transcription of the MAP kinase3 gene that is known to be involved in stress responses. This study shows that higher plants, although bereft of purinoceptor homologues, could have evolved a distinct mechanism to transduce the ATP signal at the plasma membrane.

. 11. Complementary DNAs encoding the short forms of G␣ s WT and G␣ s * (9) were subcloned into pUAST (7) and used to transform flies [A. Spradling, in Drosophila: A Practical Approach, D. Roberts, Ed. (IRL Press, Oxford, 1986), pp. 75 -197]. The UAS-G␣ s lines were verified by Msc I digestion of polymerase chain reaction-amplified G␣ s transgenes to detect the presence of a site found in G␣ s WT but lacking as a result of the Q215L mutation. 12. 238Y and 201Y are described in (17) and C232 in (18). C747 shows identical expression to that of C772 (17) (24). C309 is described by J. D. Armstrong [thesis, University of Glasgow, Scotland (1995)]. The O'Kane laboratory screened 500 P-GAL4 lines to identify OK66, OK86, OK62, OK107, OK348, and OK415. 13. T. Tully and W. Quinn, J. Comp. Physiol. 157, 263 (1985); T. Tully, T. Preat, S. Boynton, M. Del Vecchio, Cell 79, 35 (1994). 14. In total, we analyzed behaviorally 16 P-GAL4 insertions that expressed to some degree in MBs. Flies with four insertions (OK66, OK86, KL65, and KL107) showed reduced learning in the absence of expression of constitutively activated G␣ s *, most likely because of genetic background differences. These were not tested further. Flies with eight insertions showed defects in olfactory acuity (30Y, C35, OK62, OK107, C302, OK415, and C532) or shock reactivity (C772), as transheterozygotes with UAS-G␣ s *. These were eliminated from further behavioral testing. Flies with the remaining four P-GAL4 insertions (201Y, 238Y, C309, and C747) showed disrupted learning but normal olfactory acuity and shock reactivity, as transheterozygotes with at least one UAS-G␣ s * insertion. We characterized a total of two P-GAL4 insertions (C232 and OK348) that expressed to some degree in the CC. 15. We suggest that olfactory learning results from G sdependent convergence of the conditional stimulus (CS) and unconditional stimulus (US) in the MBs. This belief is reinforced by the observation that expression of G␣ s * in the CC does not affect learning.

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