The effects of nano-TiO2 (rutile) on the photochemical reaction of chloroplasts of spinach were studied. The results showed that when spinach was treated with 0.25% nano-TiO2, the Hill reaction, such as the reduction rate of FeCy, and the rate of evolution oxygen of chloroplasts was accelerated and noncyclic photophosphorylation (nc-PSP) activity of chloroplasts was higher than cyclic photophosphorylation (c-PSP) activity, the chloroplast coupling was improved and activities of Mg2+-ATPase and chloroplast coupling factor I (CF1)-ATPase on the thylakoid membranes were obviously activated. It suggested that photosynthesis promoted by nano-TiO2 might be related to activation of photochemical reaction of chloroplasts of spinach.

Previous research showed that nano-TiO2 could significantly promote photosynthesis and greatly improve growth of spinach, but we also speculated that an increase of spinach growth by nano-TiO2 treatment might be closely related to the change of nitrogen metabolism. The effects of nanoanatase TiO2 on the nitrogen metabolism of growing spinach were studied by treating them with nano-anatase TiO2. The results showed that nano-anatase TiO2 treatment could obviously increase the activities of nitrate reductase, glutamate dehydrogenase, glutamine synthase, and glutamic-pyruvic transaminase during the growing stage. Nano-anatase TiO2 treatment could also promote spinach to absorb nitrate, accelerate inorganic nitrogen (such as NO3--N and NH4+-N) to be translated into organic nitrogen (such as protein and chlorophyll), and enhance the fresh weight and dry weights.

Having a photocatalyzed characteristic, 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. By electrophoresis and Western blot methods, the results of the experiments proved that Rubisco from the nano-anatase TiO2-treated spinach during the extraction procedure of Rubisco was found to consist of Rubisco and a heavier molecular-mass protein (about 1,200 kDa) comprising both Rubisco and Rubisco activase. 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. It suggested that the mechanism of nano-anatase TiO2 activating Rubisco of spinach was that the complex of Rubsico and Rubisco activase was induced in spinach, which promoted Rubsico carboxylation and increased the rate of photosynthetic carbon reaction.

The improvement of spinach growth is proved to relate to N2 fixation by nano-anatase TiO2 in this study. The results show that all spinach leaves kept green by nano-anatase TiO2 treatment and all old leaves of control turned yellow white under culture with N-deficient solution. And the fresh weight, dry weight, and contents of total nitrogen, NH4(+), chlorophyll, and protein of spinach by nano-anatase TiO2 treatment presented obvious enhancement compared with control. Whereas the improvements of yield of spinach were not as good as nano-anatase TiO2 treatment under N-deficient condition, confirming that nano-anatase TiO2 on exposure to sunlight could chemisorb N2 directly or reduce N2 to NH3 in the spinach leaves, transforming into organic nitrogen and improving the growth of spinach. Bulk TiO2 effect, however, was not as significant as nano-anatase TiO2. A possible metabolism of the function of nano-anatase TiO2 reducing N2 to NH3 was discussed.

The effects of nano-TiO2 (rutile) on the chloroplast aging of spinach under light were studied. The results showed that when the chloroplasts were illuminated for 1, 5, and 10 min with 500 micromol/cm2/min light intensity, respectively, the evolution oxygen rate was rapidly increased; when the chloroplasts were treated for 20, 30, and 40 min with 500 micromol/cm2/min light intensity, respectively, the evolution oxygen rate was gradually decreased. While spinach was treated with 0.25% nano-TiO2, the rate of evolution oxygen of chloroplasts in different illumination times (1, 5, 10, 20, 30, and 40 min) was higher than that of control, and when the illumination time was over 10 min, the reduction of the evolution oxygen rate was lower than that of control. It suggested that nano-TiO2 treatment could protect chloroplasts from aging for long-time illumination. The mechanism researches indicated that nano-TiO2 treatment could significantly increase the activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), decrease accumulation of reactive oxygen free radicals and the level of malondialdehyde (MDA), and maintain stability of membrane structure of chloroplast under light.