Inhibitive Mechanisms Of Lead Ion On Photosynthesis Of Spinach

Heavy metal pollution is a significant stress factor to all the component of plant growth, especially to the photosynthesis of plant. There are many researches had proved that heavy metal pollution had inhibitions on pigments of photosynthesis, photosynthetic tissue, the course of photosynthesis and photosynthetic rate etc. But the mechanisms of Pb²⁺ on the inhibitions of photosynthesis are still unclear. Given this we choose the Pb²⁺ treated spinach as experimental material to study the inhibitive effects of Pb²⁺ on photosynthesis. Six main aspects are involved.(1)The effects of Pb²⁺ on energy distribution and photochemical activity of spinach chloroplast were studied. The experimental results showed that the absorption peak intensity of chloroplast obviously decreased in red and blue region and produced optical flattering; fluorescence quantum yield nearby 680 nm of chloroplast greatly declined; the excitation band nearby 440 nm of chloroplast significantly descended; Pb²⁺ treatments reduced of the rate of whole chain electron transport, photochemical activities of PSII DCPIP photoreduction and oxygen evolution, but the photoreduction activities of PSI were little changed. Together, the studies of the experiments showed that Pb²⁺ decreased absorption of light on spinach chloroplast and inhibited excitation energy to be absorbed by LHCâ…¡and transferred to PSâ…¡, then reduced the conversion from light energy to electron energy, and decelerated electron transport, water photolysis and oxygen evolution.(2)The effects of Pb²⁺ on energy transfer within photosystemâ…¡and oxygen evolution Lead were studied. The experimental results showed that PSâ…¡microenvironment was damaged and absorbance for visible light decreased; energy transfer among amino acids within PSâ…¡protein complex was inhibited and energy transport from tyrosine residue to chlorophyll a was declined; photochemistry activity and oxygen evolving rate of PSâ…¡was reduced.(3)The effects of Pb²⁺on activities of photochemical reaction and key enzymes of carbon assimilation in spinach chloroplast were studied. The results showed that Pb²⁺ treatment could significantly inhibit the Hill reaction activity of spinach chloroplast and photophosphorylation, and it had a more conspicuous effect on cyclic photophosphorylation than non-cyclic photophosphorylation. The activities of ATPase on the thylakoid membrane were severely inhibited under Pb²⁺ treated condition, and Ca2+ -ATPase activity was affected more obviously than Mg2+-ATPase activity. Meanwhile, the activities of the key enzymes of carbon assimilation were also significantly reduced by Pb²⁺, especially Rubisco activase.(4)The effects of Pb²⁺ on the Kinetic and Spectral Characterization of Rubisco activase were studied. The Rubisco was purified from spinach. It showed that UV-visible absorption spectrum of Pb²⁺ treatment Rubisco of was the same as the control, however, the absorbency intensity at 211 nm and 280 nm were gradually enhanced by the increase of Pb²⁺ concentration. Fluorescence spectra exhibited that Pb²⁺ addition did not alter fluorescence emission spectra of Rubisco though the intensity was decreased. The enzyme activity assays showed that the reaction between Rubisco and Pb²⁺ was second-order, which means that the decrease of Rubisco activity was accelerated by a low concentration of Pb²⁺ and slowed by a high concentration of Pb²⁺. By UV absorption and fluorescence spectroscopy assays, the Pb²⁺ was determined to be directly bound to Rubisco; Circular dichroism (CD) spectra proved that the random coil,β-sheet andβ-turn contents of Rubisco treated with Pb²⁺ are significantly increased, and theα-helix content is sharply decreased in comparison to those of the control, showing that various concentrations of Pb²⁺ result in the secondary structure of Rubisco being severely destroyed, and the activity of Rubisco lost.(5)Spinach were cultured with different concentrations of Pb²⁺, then Chloroplasts of spinach were assayed for overproduction of reactive oxygen species (ROS) such as superoxide radicals (O2·–), hydrogen peoxide (H2O2), and of lipid peroxide (malonyldialdehyde, MDA). The experimental results showed that with increasing concentration of Pb²⁺ the activities of the antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and glutathione content, oxygen-evolving rate, chlorophyll content. Increase of both ROS and lipid peroxide content, and reduction of photosynthesis and activities of the antioxidant defense system indicated that spinach chloroplast underwent a stress condition due to an oxidative attack. Seedling growth cultivated in containing Pb²⁺ media was significantly inhibited. The results imply that spinach chloroplast was not able to tolerate the oxidative stress induced by Pb²⁺ due to having no an effective antioxidant defense mechanism.(6)The effects of different concentrations of Pb²⁺ on the nitrogen metabolism of growing spinach were studied. The experimental results showed that with increasing concentration of Pb²⁺ the absorption of nitrate-nitrogen by spinach decreased, and the activities of nitrate reductase, glutamate dehydrogenase, glutamine synthase, and glutamic–pyruvic transaminase presented a significant reduction. The accumulation of ammonium nitrogen in cell was increased with increasing concentration of Pb²⁺, and the synthesis of organic nitrogen compounds such as protein and chlorophyll and so on was inhibited, suggesting that Pb²⁺ could inhibit inorganic nitrogen to be translated into organic nitrogen in spinach.