Ferredoxin/thioredoxin system plays an important role in the chloroplastic NADP status of Arabidopsis

Abstract

NADP is a key electron carrier for a broad spectrum of redox reactions, including photosynthesis. Hence, chloroplastic NADP status, as represented by redox status (ratio of NADPH to NADP ) and pool size (sum of NADPH and NADP ), is critical for homeostasis in photosynthetic cells. However, the mechanisms and molecules that regulate NADP status in chloroplasts remain largely unknown. We have now characterized an Arabidopsis mutant with imbalanced NADP status (inap1), which exhibits a high NADPH/NADP ratio and large NADP pool size. inap1 is a point mutation in At2g04700, which encodes the catalytic subunit of ferredoxin/thioredoxin reductase. Upon illumination, inap1 demonstrated earlier increases in NADP pool size than the wild type did. The mutated enzyme was also found in vitro to inefficiently reduce m-type thioredoxin, which activates Calvin cycle enzymes, and NADP-dependent malate dehydrogenase to export reducing power to the cytosol. Accordingly, Calvin cycle metabolites and amino acids diminished in inap1 plants. In addition, inap1 plants barely activate NADP-malate dehydrogenase, and have an altered redox balance between the chloroplast and cytosol, resulting in inefficient nitrate reduction. Finally, mutants deficient in m-type thioredoxin exhibited similar light-dependent NADP dynamics as inap1. Collectively, the data suggest that defects in ferredoxin/thioredoxin reductase and m-type thioredoxin decrease the consumption of NADPH, leading to a high NADPH/NADP ratio and large NADP pool size. The data also suggest that the fate of NADPH is an important influence on NADP pool size.

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

“…De novo synthesis of NADP + in cytosol, chloroplasts, and peroxisomes is carried out by the ATP-dependent NAD kinases: NADK1, NADK2, and NADK3 43 , among which NADK2 in chloroplasts, is a light-dependent enzyme 44 . Illumination of 3week-old plants for 5 min to 1 h gradually increases the NADP + and NADPH pools in the rosette leaves of WT plants but not in a nadk2 mutant, indicating that, under illumination, the synthesis of NADP + from NAD + by NADK2 in the chloroplasts is the major NADP + synthesis pathway.…”

“…Illumination of 3week-old plants for 5 min to 1 h gradually increases the NADP + and NADPH pools in the rosette leaves of WT plants but not in a nadk2 mutant, indicating that, under illumination, the synthesis of NADP + from NAD + by NADK2 in the chloroplasts is the major NADP + synthesis pathway. The newly synthesized NADP + is then reduced to NADPH by FNR, and the NADPH/NADP + ratio is maintained at~1 in the first 5 min of illumination, and gradually decreases to~0.7 after 60 min of illumination 44 .…”

Computational simulation of the reactive oxygen species and redox network in the regulation of chloroplast metabolism

“…TRX-f1 and TRX-f2 function in activation of Calvin-Benson-Bassham cycle (CBB) enzymes and γ-subunit of F-ATP synthase [4]. TRX-m1, -m2, -m3 and-m4 are suggested to regulate targets which control the NADPH/NADP ratio [5] which is linked to their ability to efficiently activate the NADPH-dependent malate dehydrogenase [6]. TRX-x, NTRC and TRX-lilium1 (ACHT4a) were identified as reductants of the 2-cysteine peroxiredoxin (2CysPRX) [7][8][9], and TRX-y1 and-y2 as reductant of PRX-Q [10].…”

Computational simulation of the reactive oxygen species and redox network in the regulation of chloroplast metabolism

“…TRX-f1 and TRX-f2 function in activation of Calvin-Benson-Cycle (CBC) enzymes and γ-subunit of F-ATP synthase [4]. TRX-m1, -m2, -m3 and -m4 are suggested to regulate targets which control the NADPH/NADP ratio [5] which is linked to their ability to efficiently activate the NADPH-dependent malate dehydrogenase [6]. TRX-x, NTRC and TRX-lilium1(ACHT4a) were identified as reductants of the 2-cysteine peroxiredoxin (2-CysPRX) [7–9], and TRX-y1 and –y2 as reductant of PRX-Q [10].…”