LONG-TERM CHILLING OF YOUNG TOMATO PLANTS UNDER LOW-LIGHT .5. KINETICAND MOLECULAR-PROPERTIES OF 2 KEY ENZYMES OF THE CALVIN CYCLE IN LYCOPERSICON-ESCULENTUM MILL AND LYCOPERSICON-PERUVIANUM MILL

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 (Lycopersic on esculentum Mill.) and in four lines of a wild tomato (L. peruvianum Mill.) from different altitudes. During chilling for 14 d at 10 degre es C and low light, the activation energy (E(A)) of the reaction catal yzed 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 lo ss or only small losses of enzyme activity were observed during the ch illing. Together with the change in E(A), this indicates that the latt er species is able to acclimate its Calvin-cycle enzymes to low temper atures. In L. esculentum, the chilling stress resulted in the irrevers ible loss of 57% of the initial sFBPase activity. Under moderately pho toinhibiting chilling conditions for 3 d, the L. peruvianum line from an intermediate altitude showed the largest decreases in both the rati o 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 p recipitation and gel filtration and characterized by two-dimensional e lectrophoresis. The enzyme from L. esculentum had three isoforms of th e small subunit of Rubisco, each with different isoelectric points. Of these, the L. peruvianum enzyme contained only the two more-acidic is oforms. Arrhenius plots of the specific activity of purified Rubisco s howed 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 altitud es was unaffected by chilling. We tested several possibile explanation s for Rubisco inactivation, using two-dimensional electrophoresis, ana lytical ultracentrifugation, gel filtration and inhibitor tests. No in dications were found for differential expression of the subunit isofor ms, proteolysis, aggregation, subunit disassembly, or inhibitor accumu lation in the enzyme from chilled L. esculentum. We suggest that the a ctivity loss in the L. esculentum enzyme upon chilling is the result o f a modification of sulfhydryl groups or other sidechains of the prote in.