Photoprotection Mediated By Mitochondrial Respiratory Electron Transport And Its Regulation Mechanism In Rumex K-1 Leaves

Excess light energy is harmful to plants and leads to photoinhibition. Since most plants cannot escape exposure to excess light, they have evolved defense systems that dissipate excess light energy. These systems include the thermal dissipation of light energy in pigment-protein complexes in the light-harvesting antennae, the cyclic electron flow around PSI and the water-water cycle. Though such intra-chloroplastic defense mechanisms have been studied extensively, little is known about the extra-chloroplastic defense mechanism. It has been presented that excess reducing equivalents generated in chloroplasts can be transported to mitochondria via shuttle machineries, and oxidized by the alternative oxidase (AOX) pathway. Therefore, it has been speculated that the AOX pathway might have a particular role in protection of plants from photoinhibition, but direct evidence for the role of AOX as a mechanism in protecting plants against photoinhibition is still limited. In this study, the physiological function of the AOX pathway in photoprotection of plants was confirmed in Rumex K-1 leaves. And the mechanism of up-regulation of AOX pathway by light was discussed. The main results obtained are as follows:(1) A significant decrease in the rate of CO₂ assimilation in salicylhydroxamic acid (SHAM)-treated Rumex K-1 leaves was observed over a range of different light intensities. Gas exchange data further revealed that stomatal conductance was not significantly affected and the internal CO₂ concentration in Rumex K-1 leaves was enhanced by the inhibition of AOX pathway, suggesting that a reduction in stomatal density or aperture size was not responsible to the decreased photosynthetic rate. Furthermore, a reduction in Calvin cycle capacity and photochemical efficiency were not responsible to the decreased assimilation rate. And we measured photosynthetic rate under high concentrations of CO₂ conditions in which the oxygenase activity of Rubisco is minimized and photorespiration is not active. The photosynthetic rate in SHAM-treated leaves is not significantly different from than in the control leaves under non-photorespiratory conditions, strongly suggesting that the decrease in assimilation rate under ambient atmospheric conditions is linked to the photorespiratory restriction.A possible link between mitochondrial coupling state and photosynthesis is the requirement for rapid oxidation of NADH produced in the mitochondrion during conversion of photorespiratory glycine to serine. A dramatic decrease in the rate of conversion of glycine to serine was observed when the AOX pathway was inhibited. These data demonstrate that the inhibition of AOX pathway restricts photorespiratory flux. The decrease in photosynthesis due to the inhibition of AOX pathway may be associated with a limitation in ribulose-1,5-bisphosphate regeneration in the Calvin cycle due to reduced glycollate-2-P recycling into glycerate-3-P via the photorespiratory pathway. Furthermore, glycine has been shown to accumulate in the light when AOX pathway was inhibited. This very high level of glycine could result in an accumulation of glyoxylate which was shown to inhibit photosynthesis by reducing the activation state of Rubisco.(2) The observation that activities of malate-oxaloacetate shuttle and AOX pathway increased obviously under high light in Rumex K-1 leaves indicates that excess reducing equivalents generated by photosynthesis were transported from chloroplasts to mitochondria and oxidized by AOX pathway. Inhibition of AOX pathway by SHAM in Rumex K-1 leaves decreased the activity of malate-oxaloacetate shuttle, causing over-reduction of PSâ…¡acceptor side and the decrease in total driving forces for photosynthetic electron transport (DF_ABS) because of accumulation of excess reducing equivalents in chloroplasts. The decrease in total driving forces for photosynthetic electron transport (DF_ABS) restricted photosynthetic linear electron flow (ETR), which inevitably limited of generation of pH gradient across thylakoid and decreased the de-epoxidation of xanthophyll cycle pigments indicated by decrease inΔPRI. Therefore, formation of NPQ was suppressed due to the inhibition of AOX pathway. Moreover, effect of inhibition of AOX pathway on NPQ formation was lesser at higher CO₂ supply (20mM NaHCO₃) than that at lower CO₂ supply (1mM NaHCO₃). Therefore, AOX pathway plays an essential role in formation of NPQ under high light via the generation of pH gradient across thylakoid, protecting photosynthetic apparatus against photodamage.(3) The inhibition of AOX pathway by SHAM decreasedΦ_PSâ…¡ and the O₂ evolution rate, and increased non-Q_B reducing reaction center, causing more severe photoinhibition even under low light in Rumex K-1 leaves. Under low light, the loss of the function of AOX pathway was compensated by up-regulation of other photoprotection pathways such as cyclic electron transport around PSI, water-water cycle and antioxidant enzymes, , which alleviated the accumulation of reactive oxygen species (ROS) when AOX pathway was inhibited. But under high light, it was not able to compensate the loss of the function of AOX pathway by the other photoprotection pathways when the AOX pathway was inhibited, leading to more severe accumulation of ROS. This result indicates that other photoprotection pathways were able to partially replace the function of AOX pathway under low light, but not under high light. (4) Inhibition of AOX pathway decreased the maximum quantum yield for primary photochemistry (φ_Po), the excitation efficiency of electron transport beyong Q_A^- (Ψ₀) and the quantum yield of electron transport (φ_Eo), causing more severe photoinhibition under high light in Rumex K-1 leaves. However, the inhibition of AOX pathway did not change the level of photoinhibition under high light in the presence of the inhibitor of chloroplast D1 protein synthesis, chloramphenicol, indicating that the inhibition of the AOX pathway did not accelerate the photodamage to PSâ…¡directly. All of these results suggest that the AOX pathway plays an important role in the protection of plants against photoinhibition by minimiszing the inhibition of the repair of the photodamaged PSâ…¡.(5) It is noteworthy that activation state of malate-oxaloacetate shuttle increased very quickly upon irradiation in Rumex K-1 leaves, it increased to about 75% of full activation only 1min after irradiation, suggesting that malate-OAA shuttle was activated quickly to export excess reducing equivalents generated by photosynthesis to mitochondria and cytosol during photosynthetic induction. During photosynthetic induction, inhibition of AOX pathway by SHAM restricted light activation of malate-oxaloacetate shuttle, which caused over-reduction of PSI acceptor side and over-accumulation of Q_A^ˉ, thereby limited photosynthetic linear electron flow (ETR). The limitation of ETR without changing in light absorption (ABS/RC) and trapping (TR₀/RC) caused imbalance between light energy absorption and utilization during photosynthetic induction. The limitation of ETR also restricted formation of pH gradient across thylakoid indicated by decrease in de-epoxidation of xanthophyll cycle, restricting formation of non-photochemical quenching (NPQ). The imbalance between light energy absorption and utilization and the suppression of NPQ formation inevitably resulted in over-excitation of PSâ…¡reaction centres during photosynthetic induction. The induction of CO₂ assimilation was delayed by SHAM-treatment, which was reversed partly by exogenously-application of ATP, suggesting that the inhibition of AOX pathway delayed light activation of Calvin cycle enzymes due to the restriction of formation of pH gradient across thylakoid to generate ATP. Therefore, mitochondrial AOX pathway acts as a sink for electrons generated by photosynthesis, which protects photosynthetic apparatus against photoinhibition and accelerates induction of CO₂ assimilation during photosynthetic induction.(6) Light increased the capacity of AOX pathway in Rumex K-1 leaves. But the capacity of AOX pathway did not increase when the photosynthetic electron transport from Q_A to Q_B was inhibited by DCMU in the light, which suggests that light regulates the AOX pathway through photosynthetic signals. The DCMU or MV-pretreatment, which induced more severe accumulation of O₂^- and H₂O₂ and inhibited the generation of NADPH, did not enhance the capacity of the AOX pathway under light, suggesting that the accumulation of NADPH rather than ROS generated by photosynthesis was involved in light-dependent increase in AOX pathway capacity.. Exogenously-application of NADPH and OAA did not change the capacity of AOX pathway in the dark. But the capacity of AOX pathway increased with the increase of NADPH or OAA concentration in the light, which suggests that NADPH was not the direct signal in light-dependent induction of AOX pathway.Furthermore, it was observed that pyruvate content increased with the increase of light intensity in control leaves. And the pyruvate content increased with the increase of NADPH or OAA concentration in the light. Given that the excess NADPH can be transported from chloroplasts to mitochondria accompanied with formation of pyruvate, and the DCMU or MV-pretreatment did not enhance the formation of pyruvate in the light, it is reasonable to suggest that the pyruvate might play a major role in light-dependent up-regulation of AOX pathway.