The properties of two Calvin-cycle key enzymes, i.e. stromal fructose-l,6-bisphosphatase (sFBPase) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were studied in the cultivated tomato (Lycopersicon esculentum Mill.) and in four lines of a wild tomato (L. peruvianum Mill.) from different altitudes. During chilling for 14 d at 10 degrees C and low light, the activation energy (E(A)) of the reaction catalyzed by sFBPase decreased by 5-10 kJ.mol(-1) in L. esculentum and the three L. peruvianum lines from high altitudes. In L. peruvianum, no loss or only small losses of enzyme activity were observed during the chilling. Together with the change in E(A), this indicates that the latter species is able to acclimate its Calvin-cycle enzymes to low temperatures. In L. esculentum, the chilling stress resulted in the irreversible loss of 57% of the initial sFBPase activity. Under moderately photoinhibiting chilling conditions for 3 d, the L. peruvianum line from an intermediate altitude showed the largest decreases in both the ratio of variable to maximum chlorophyll fluorescence (F-v/F-m) and the in-vivo activation state of sFBPase, while the other L. peruvianum lines showed no inhibition of sFBPase activation. Ribulose-1,5-bisphosphate carboxylase/oxygenase was isolated by differential ammonium-sulfate precipitation and gel filtration and characterized by two-dimensional electrophoresis. The enzyme from L. esculentum had three isoforms of the small subunit of Rubisco, each with different isoelectric points. Of these, the L. peruvianum enzyme contained only the two more-acidic isoforms. Arrhenius plots of the specific activity of purified Rubisco showed breakpoints at approx. 17 degrees C. Upon chilling, the specific activity of the enzyme from L. esculentum decreased by 51%, while E(A) below the breakpoint temperature increased from 129 to 189 kJ.mol(-1). In contrast, Rubisco from the L. peruvianum lines from high altitudes was unaffected by chilling. We tested several possibile explanations for Rubisco inactivation, using two-dimensional electrophoresis, analytical ultracentrifugation, gel filtration and inhibitor tests. No indications were found for differential expression of the subunit isoforms, proteolysis, aggregation, subunit disassembly, or inhibitor accumulation in the enzyme from chilled L. esculentum. We suggest that the activity loss in the L. esculentum enzyme upon chilling is the result of a modification of sulfhydryl groups or other sidechains of the protein.
