In vitro reassembly of tobacco ribulose-1,5-bisphosphate carboxylase/oxygenase from fully denatured subunits.

It has been generally proved impossible to reassemble ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from fully denatured subunits in vitro in higher plant, because large subunit of fully denatured Rubisco is liable to precipitate when the denaturant is removed by common methods of direct dilution and one-step dialysis. In our experiment, the problem of precipitation was resolved by an improved gradual dialysis method, which gradually decreased the concentration of denaturant. However, fully denatured Rubisco subunits still could not be reassembled into holoenzyme using gradual dialysis unless chaperonin 60 was added. The restored activity of reassembled Rubisco was approximately 8% of natural enzyme. The quantity of reassembled Rubisco increased greatly when heat shock protein 70 was present in the reassembly process. ATP and Mg2+ were unnecessary for in vitro reassembly of Rubisco, and Mg2+ inhibited the reassembly process. The reassembly was weakened when ATP, Mg2+ and K+ existed together in the reassembly process.

Positive charges on lysine residues of the extrinsic 18 kDa protein are important to its electrostatic interaction with spinach photosystem II membranes.

To determine the contribution of charged amino acids to binding with the photosystem II complex (PSII), the amino or carboxyl groups of the extrinsic 18 kDa protein were modified with N-succinimidyl propionate (NSP) or glycine methyl ester (GME) in the presence of a water-soluble carbodiimide, respectively. Based on isoelectric point shift, 4-10 and 10-14 amino groups were modified in the presence of 2 and 4 mM NSP, respectively. Similarly, 3-4 carboxyl groups were modified by reaction with 100 mM GME. Neutralization of negatively charged carboxyl groups with GME did not alter the binding activity of the extrinsic 18 kDa protein. However, the NSP-modified 18 kDa protein, in which the positively charged amino groups had been modified to uncharged methyl esters, failed to bind with the PSII membrane in the presence of the extrinsic 23 kDa protein. This defect can not be attributed to structural or conformational alterations imposed by chemical modification, as the fluorescence and circular dichroism spectra among native, GME- and NSP-modified extrinsic 18 kDa proteins were similar. Thus, we have concluded that the positive charges of lysyl residues in the extrinsic 18 kDa protein are important for its interaction with PSII membranes in the presence of the extrinsic 23 kDa protein. Furthermore, it was found that the negative charges of carboxyl groups of this protein did not participate in binding with the extrinsic 23 kDa protein associated with PSII membranes.

Photosynthetic acclimation in rice leaves to free-air CO2 enrichment related to both ribulose-1,5-bisphosphate carboxylation limitation and ribulose-1,5-bisphosphate regeneration limitation.

Net photosynthetic rates (Pns) in leaves were compared between rice plants grown in ambient air control and free-air CO2 enrichment (FACE, about 200 micromol mol(-1) above ambient) treatment rings. When measured at the same CO2 concentration, the Pn of FACE leaves decreased significantly, indicating that photosynthetic acclimation to high CO2 occurs. Although stomatal conductance (Gs) in FACE leaves was markedly decreased, intercellular CO2 concentrations (Ci) were almost the same in FACE and ambient leaves, indicating that the photosynthetic acclimation is not caused by the decreased Gs. Furthermore, carboxylation efficiency and maximal Pn, both light and CO2-saturated Pn, were decreased in FACE leaves, as shown by the Pn-Ci curves. In addition, the soluble protein, Rubisco (ribulose-1,5-bisphosphate caboxylase/oxygenase), and its activase contents as well as the sucrose-phosphate synthase activity decreased significantly, while some soluble sugar, inorganic phosphate, chlorophyll and light-harvesting complex II (LHC II) contents increased in FACE leaves. It appears that the photosynthetic acclimation in rice leaves is related to both ribulose-1,5-bisphosphate (RuBP) carboxylation limitation and RuBP regeneration limitation.