In planta study of photosynthesis using genetically encoded NADPH and NADH/NAD⁺ fluorescent protein sensors

Abstract

Being one of the most important carriers of reducing equivalents, pyridine nucleotides are involved in many metabolic processes in plants. Several in vitro methods have been developed to measure these pyridine nucleotides level, including bioluminescence, liquid chromatography, mass spectrometry, enzymatic assay and radioactive tracer. All of these methods, however, require a complex extraction process; therefore, determination of their dynamic changes in different subcellular compartments of various cells is not possible. The recent development of fluorescent based protein biosensors in animal systems has shed new light to pyridine nucleotides in vivo measurements. In my study, two pyridine nucleotides sensors, namely iNAP (NADPH) and SoNar (NADH/NAD+) were introduced into Arabidopsis thaliana (Arabidopsis) to monitor their dynamic changes in planta. The real-time changes of pyridine nucleotides in different subcellular compartments during photosynthesis has not been observed before. In chapter 2, transgenic Arabidopsis expressing iNAP or SoNar in the cytosol, plastid stroma (TKTP) and peroxisome (SRL) were generated and characterized. The ratio shifts of these two sensors correspond to changes in NADPH level and NADH/NAD+ ratio. My work revealed that 180s light exposure at 296 μmol m−2 s−1 can induce ratiometric shifts in cytosol-iNAP1 (high affinity), TKTP-iNAP4 and iNAP4-SRL (low affinity) lines but not in cytosol-iNAP4, TKTP-iNAP1 and iNAP1-SRL lines, implying free cytosolic NADPH is lower than that in plastids and peroxisomes of 10-day-old cotyledon. Various groups have proposed that either malate-OAA shuttle or photorespiration, or both can supply reducing equivalents to mitochondrial electron transport chain (mETC) during photosynthesis. However, this has not been examined in the whole plant level. To investigate this problem, photorespiration inhibitor (aminoacetonitrile) and a high CO2 condition were applied to my sensor lines in chapter 3. At 296 μmol m−2 s−1, photorespiration supplies a large amount of reducing equivalents to mitochondria, which exceeds the NADH-dissipating capacity in mETC during photosynthesis. It was concluded that photorespiration is the major NADH contributor for mETC during photosynthesis, and the malate-OAA shuttle serves to export surplus reducing equivalents from mitochondria to cytosol. Whether guard cell (GC) photosynthesis contributes to stomatal regulation has been a matter of debate for decades. In chapter 4, illumination increases ATP, NADPH and pH level in mesophyll chloroplasts but not in GC chloroplasts (GCCs). Together with the observations that the starch level in GCC decreased in the first two hours of illumination, increased thereafter and the ability of cytosolic ATP to enter GCCs, suggesting that the main function of GCC is not photosynthesis, but to serve as a starch reservoir, which can be mobilized as sugars for promoting stomatal opening during the dawn. In summary, by employing the novel iNAP and SoNar biosensors, I investigated the dynamic changes of NADPH and NADH/NAD+ ratio induced by photosynthesis in several subcellular compartments of Arabidopsis under various conditions. Two schematic models that can advance our understandings on the physiology of mesophyll and GC during photosynthesis were generated based on my data, which show the power of biosensors as valuable tools for in planta study.