Long-term chilling of young tomato plants under low light

The properties of two Calvin-cycle key enzymes, i.e. stromal fructose-1,6-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°C and low light, the activation energy (E_A) of the reaction catalyzed by sFBPase decreased by 5–10 kJ·mol^–1 inL. esculentum and the threeL. peruvianum lines from high altitudes. InL. 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. InL. esculentum, the chilling stress resulted in the irreversible loss of 57% of the initial sFBPase activity. Under moderately photoinhibiting chilling conditions for 3 d, theL. 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 otherL. 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 fromL. esculentum had three isoforms of the small subunit of Rubisco, each with different isoelectric points. Of these, theL. peruvianum enzyme contained only the two more-acidic isoforms. Arrhenius plots of the specific activity of purified Rubisco showed breakpoints at approx. 17°C. Upon chilling, the specific activity of the enzyme fromL. esculentum decreased by 51%, while E_A below the breakpoint temperature increased from 129 to 189 kJ·mol^–1. In contrast, Rubisco from theL. 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 chilledL. esculentum. We suggest that the activity loss in theL. esculentum enzyme upon chilling is the result of a modification of sulfhydryl groups or other sidechains of the protein.Abbreviations a.s.l. above sea level - Chl chlorophyll - DTT dithiothreitol - E_A activation energy - FBP fructose-1,6-bisphosphate - F_v/F_m ratio of variable to maximum chlorophyll fluorescence - HL high light (500 mol photons·m^–2·s^–1) - LSU large subunit of Rubisco - ME 2-mercaptoethanol - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - sFBPase stromal fructose-1,6-bisphosphatase - SSU small subunit of Rubisco