Energy production in chloroplast and mitochondrion

Abstract

In the form of adenosine triphosphate (ATP), energy is constitutively spent in living organisms for many metabolic activities, such as growth, defenses, and homeostasis. In plants, ATP is primarily generated from two double membrane enclosed organelles, chloroplast and mitochondrion, by the process of photosynthesis and respiration respectively. In my works, I applied different molecular tools to investigate the relationship of chloroplasts and mitochondria in term of energy metabolism using model plant, Arabidopsis thaliana. The techniques used in this study include FRET-based visualization of ATP in vivo using ATP-specific fluorescent probe and confocal microcopy. By introducing the probe into cell compartments (cytosol and plastid), this technique allows me to monitor the dynamic of ATP in these compartments in a non-destructive way. Here, my works show that the demand of ATP in chloroplasts is vigorous whereas mitochondria play a major role in supplying ATP to cytosol. Depletion of cytosolic ATP was observed when Complex III or ATP synthase in mitochondria were inhibited. In cotyledon, ATP concentration in chloroplasts is always low comparing to the cytosol, suggesting that the transfer of ATP between these two compartments is inefficient. Upon illumination, ATP concentration in stroma increased instantly due to photosynthesis activity, but stromal ATP was rapidly consumed by the metabolic activities in chloroplasts when light was withdrawn. The dependency between mitochondria and chloroplasts was navigated by measuring their performance when their corresponding partner was inhibited. Inhibition on mitochondrial compartments had lower the ATP production capacity in chloroplasts, implying that functional mitochondrial activity is required for optimum photosynthesis activity. This finding is supported by results obtained from alternative approaches such as chlorophyll fluorescence analysis and oxygen evolution measurement. On the other hand, photosynthesis also influences mitochondrial activity. Here, the mitochondrial activity was examined by in vivo pH-specific FRET sensor, mt-cpYFP, targeted to mitochondrial matrix compartment. Mitochondrial matrix pH was elevated upon illumination, suggesting that mitochondrial electron transfer activity was boosted by photosynthesis activity. ATP concentration also increased in cytosol during illumination, implying enhanced ATP production in mitochondria. The contribution of cyclic electron flow (CEF) in stroma ATP production was also explored by examining the CEF-deficient mutant line, pgr5. Application of ATP and pH probes in chloroplast stroma revealed that the photosynthesis performance of pgr5 was greatly affected in high light condition. These findings suggest that CEF is crucial in the photo-protective mechanism. Transgenic line with higher ATP and sucrose level, AtPAP2 overexpression line (OE) was studied. TEM and proteomic analysis revealed that the chloroplast composition was altered in the OE line. Moreover, increased rate of electron transfer and carbon fixation were detected in the OE chloroplast. Evaluation on the OE mitochondria showed that the mitochondrial activity in the OE line was higher than WT during illumination. In summary, increased rate of linear electron flow through OE photosynthetic apparatus generates excess reducing power and fuels the OE mitochondria for more ATP production. This spares more carbon and ATP for sucrose synthesis in the OE line.