| Having a photocatalyzed characteristics, our previous research had proved that nano-anatase TiO2 is closely related to the photosynthesis of spinach. It could not only improve the light absorbance and the transformation from light energy to electron energy and to active chemical energy but also promote carbon dioxide (CO2) assimilation of spinach. However, the mechanism of carbon reaction promoted by nano-anatase TiO2 remains largely unclear. So we choose the nano-anatase TiO2 treated spinach as experimental material to study the effects of nano-anatase TiO2 on Rubisco(ribulose-1,5-bisphosphate carboxylase/oxygenase)and its molecular chaperones– Rubisco activase, which is the most important enzyme in photosynthetic carbon reaction. Three main aspects are involved.1) RT-PCR, Northern-Blotting, polyacrylamide gel electrophoresis and Western-Blotting were carried out to study the effects of nano-anatase TiO2 treatment on the mRNA amounts and protein expression of Rubisco and Rubisco activase. It showed that the amounts of Rubisco activase (rca) mRNA in the nano-anatase TiO2 treated spinach were increased by about 51%, while bulk TiO2 (non-nano-TiO2) treatment produced an increase of only 5%. Accordingly, the protein expression of Rubisco activase from the nano-anatase TiO2 treated spinach was increased by 42% compared with control, however, bulk TiO2 treatment resulted in a 5% improvement. The amounts of Rubisco small subunit (rbcL)mRNA and Rubisco large subunit (rbcS)mRNA which compose of the Rubisco in spinach, with the nano-anatase TiO2 treatment were also increased by about 32% and 23%, respectively, and the protein expression of Rubisco from the nano-anatase TiO2 treated spinach was increased by 40% compared with control.2) The effects of nano-anatase TiO2 treatment on the structure and activity of Rubisco activase were studied. The results showed that the activity of Rubiso activase was obviously enhanced, by up to 2.5 times compared with the control, while the effects of bulk TiO2 treatment were not so significant. Spectroscopy analysis indicated that the intensities of absorption and florescence of purified Rubiso activase from the nano-anatase TiO2 treated spinach were significantly higher than that of control, the secondary structure of the purified enzyme was also very different, theα-helix,β-sheet andβ-turn contents were markedly increased, and the random coil contents were greatly decreased, proved that the secondary structure of Rubiso activase was really changed by the nano-anatase TiO2 treatment.3) It was reported first that the super-complex of Rubisco and Rubisco activase was found from the nano-anatase TiO2 treated spinach in vivo. The SDS-PAGE results showed that the purified protein from nano-anatase TiO2 treated spinach comprises of 2"unexpected"isoforms at 41 and 45 kD, which was supposed to the isoforms of Rubisco activase, and the subunits of Rubisco at 52 and 14 kD as well. The Native-PAGE results of the purified protein from nano-anatase TiO2 treated spinach suggested a heavier protein was concomitant with the Rubisco holoenzyme, whose molecular mass was estimated to about 1200 kD, which was tested to be the the super-complex of Rubisco and Rubisco activase by Western-blotting. The Rubisco carboxylase activity was 2.67 times that of Rubisco from the control and it could hydrolyze ATP in the same manner as Rubisco activase. The total sulfhydryl groups and available sulfhydryl groups of the Rubisco were 32–SH and 21–SH per mole of enzyme more than those of the Rubisco purified from the control, respectively. The circular dichroism spectra showed that the secondary structure of Rubisco from the nano-anatase TiO2-treated spinach was very different from Rubisco of the control.Accordingly, it suggestes that one of the molecular mechanisms of carbon reaction promoted by nano-anatase TiO2 is that the nano-anatase TiO2 results in the enhancement of rca, rbcL and rbcS mRNA amounts, the protein expression and activity of Rubisco activase and Rubisco, thereby, inducing the complex of Rubsico and Rubisco activase in spinach, which promoted Rubsico carboxylation and increased the rate of photosynthetic carbon reaction.
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