Adjustment of light-responsive NADP dynamics in chloroplasts by stromal pH.

Cyclic electron transfer (CET) predominates when NADP+ is at basal levels, early in photosynthetic induction; however, the mechanism underlying the subsequent supply of NADP+ to fully drive steady-state linear electron transfer remains unclear. Here, we investigated whether CET is involved in de novo NADP+ supply in Arabidopsis thaliana and measured chloroplastic NADP dynamics to evaluate responsiveness to variable light, photochemical inhibitors, darkness, and CET activity. The sum of oxidized and reduced forms shows that levels of NADP and NAD increase and decrease, respectively, in response to light; levels of NADP and NAD decrease and increase in the dark, respectively. Moreover, consistent with the pH change in the stroma, the pH preference of chloroplast NAD+ phosphorylation and NADP+ dephosphorylation is alkaline and weakly acidic, respectively. Furthermore, CET is correlated with upregulation of light-responsive NADP level increases and downregulation of dark-responsive NADP level reductions. These findings are consistent with CET helping to regulate NADP pool size via stromal pH regulation under fluctuating light conditions.

Loss of chloroplast-localized NAD kinase causes ROS stress in Arabidopsis thaliana.

Chloroplast-localized NAD kinase (NADK2) is responsible for the production of NADP+, which is an electron acceptor in the linear electron flow of photosynthesis. The Arabidopsis T-DNA-inserted mutant of NADK2 (nadk2) showed delayed growth and pale-green leaves under continuous light conditions. Under short-day conditions (8 h light / 16 h dark), the nadk2 mutant showed more severe growth inhibition.The genomic fragment containing the promoter and coding region of NADK2 complemented the phenotypes of nadk2 obtained under continuous light and short-day conditions. The nadk2 mutant produced higher amounts of H2O2 and O2-, which were reduced in the complementary line. Under short-day conditions, the nadk2 mutant accumulated more H2O2 than under continuous light conditions. The accumulation of ascorbate and up-regulation of the PDF1.2 and PR1 genes indicated that the nadk2 mutant is under ROS stress and responding to keep its living activities.

Altered metabolism of chloroplastic NAD kinase-overexpressing Arabidopsis in response to magnesium sulfate supplementation.

Nicotinamide adenine dinucleotide (NAD)/NAD phosphate (NADPH) is essential for numerous redox reactions and serve as co-factors in multiple metabolic processes in all organisms. NAD kinase (NADK) is an enzyme involved in the synthesis of NADP+ from NAD+ and ATP. Arabidopsis NADK2 (AtNADK2) is a chloroplast-localizing enzyme that provides recipients of reducing power in photosynthetic electron transfer. When Arabidopsis plants were grown on MS medium supplemented with 5 mM MgSO4, an AtNADK2-overexpressing line exhibited higher glutathione and total sulfur accumulation than control plants. Metabolomic analysis of major amino acids and organic acids using capillary electrophoresis-mass spectrometry demonstrated that overexpression of AtNADK2 affected a range of metabolic processes in response to MgSO4 supplementation.

Measurement of Chloroplastic NAD Kinase Activity and Whole Tissue NAD Kinase Assay.

Nicotinamide adenine dinucleotide phosphate (NADP) synthesis requires nicotinamide adenine dinucleotide (NAD) kinase activity, substrate NAD and ATP. The NAD kinase responds to various environmental stimuli and its activity is regulated via various regulatory pathways, such as Ca2+-dependent and redox-dependent signals. Conventional in vitro NAD kinase assay has been useful to evaluate enzyme activity; however, recent reports revealed a dynamics of NADP pool (the sum of NADP+ and NADPH) under fluctuating light condition, indicating that the rate of NADP synthesis is not always determined by NAD kinase activity. Here, we developed a novel method for the estimation of chloroplastic NAD kinase activity by quantifying the changes in the NADP amounts in response to illumination. As our approach does not involve protein extraction, it saves time (compared to the in vitro assay), thereby allowing for a sequence of assays, and provides several clues in the investigation of regulatory mechanisms behind NADP synthesis under various environmental conditions.

Inter-Organelle NAD Metabolism Underpinning Light Responsive NADP Dynamics in Plants.

Upon illumination, photosystem I in chloroplasts catalyzes light-driven electron transport from plastocyanin to ferredoxin, followed by the reduction of NADP+ to NADPH by ferredoxin:NADP+ reductase for CO2 fixation. At the beginning of photosynthesis, NADP+ supply control is dominated by de novo NADP+ synthesis rather than being recycled from the Calvin cycle. Importantly, ferredoxin distributes electrons to NADP+ as well as to thioredoxins for light-dependent regulatory mechanisms, to cyclic electron flow for more adenosine triphosphate (ATP) production, and to several metabolites for reductive reactions. We previously demonstrated that the NADP+ synthesis activity and the amount of the NADP pool size, namely the sum of NADP+ and NADPH, varies depending on the light conditions and the ferredoxin-thioredoxin system. In addition, the regulatory mechanism of cytoplasmic NAD+ supply is also involved in the chloroplastic NADP+ supply control because NAD+ is an essential precursor for NADP+ synthesis. In this mini-review, we summarize the most recent advances on our understanding of the regulatory mechanisms of NADP+ production, focusing on the interactions, crosstalk, and co-regulation between chloroplasts and the cytoplasm at the level of NAD+ metabolism and molecular transport.

Assessment of gamma radiation from a limited area of forest floor using a cumulative personal dosimeter.

To elucidate long term changes in gamma radiation from a limited region of interest of the forest floor, a simple monitoring procedure using a cumulative personal dosimeter (D-shuttle) was examined from 2016 to 2017. The test site was in a small forest in Abiko, Japan, where the initial radiocesium contamination from the Fukushima Dai-ichi Nuclear Power Plant was 60-100 kBq m-2. Three experimental plots basically containing a set of two 5 × 5 m2 observation areas were arranged at the site. The litterfall and decomposing organic layer of one area (D: decontaminated) were fully eliminated before the monitoring, whereas the other area (N: natural) was left unchanged. Five D-shuttle sets (i.e., D-shuttle, lead shield, and holder) per area were set up. One D-shuttle set could monitor the specific gamma radiation from radiocesium distributed within a limited area of ground (0.5 m radius of circle = ca. 0.8 m2 area of flat ground). The results indicated significant differences in the accumulated doses among each of the plots and areas, reflecting their soil radiocesium inventories. Interestingly, every index decreased with time, but the decreases were slower than the theoretical decay of radiocesium (134Cs and 137Cs). In addition, the accumulated dose decreased during heavy rainfall events. One possible explanation for these changes of the accumulated dose is a combination of meteorological and tree phenological phenomena, such as radiocesium from the forest canopy being newly added to the floor primarily by litterfall and soil moisture content disturbing radiation emitted from soils. This simple procedure enables long-term observation of gamma radiation from a limited area of forest floor non-invasively and semi-quantitatively.

Detection of Disulfides in Protein Extracts ofArabidopsis thaliana using Monobromobimane (mBB).

Thiol-disulfide exchange is a key posttranslational modification, determining the folding process of intra- and inter-protein structures. Thiols can be detected by colorimetric reagents, which are stoichiometrically reduced by free thiols, and by fluorescent adducts, showing fluorescence only after thioester formation. We adapted a simple three-step method for detection of disulfide bonds in proteins. After irreversible blocking of protein thiols, disulfide bonds are reduced, followed by the detection of thiols. The approach presented here provides an economical procedure that can be used to obtain a global overview of the thiol-disulfide status of proteins in plants. This method allows the detection of modifications in samples on a gel and can be used for semi-quantitative analysis.

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

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.

Increased Rate of NAD Metabolism Shortens Plant Longevity by Accelerating Developmental Senescence in Arabidopsis.

NAD is a well-known co-enzyme that mediates hundreds of redox reactions and is the basis of various processes regulating cell responses to different environmental and developmental cues. The regulatory mechanism that determines the amount of cellular NAD and the rate of NAD metabolism remains unclear. We created Arabidopsis thaliana plants overexpressing the NAD synthase (NADS) gene that participates in the final step of NAD biosynthesis. NADS overexpression enhanced the activity of NAD biosynthesis but not the amounts of NAD+, NADH, NADP+ or NADPH. However, the amounts of some intermediates were elevated, suggesting that NAD metabolism increased. The NAD redox state was greatly facilitated by an imbalance between NAD generation and degradation in response to bolting. Metabolite profiling and transcriptional analysis revealed that the drastic modulation of NAD redox homeostasis increased tricarboxylic acid flux, causing the ectopic generation of reactive oxygen species. Vascular bundles suffered from oxidative stress, leading to a malfunction in amino acid and organic acid transportation that caused early wilting of the flower stalk and shortened plant longevity, probably due to malnutrition. We concluded that the mechanism regulating the balance between NAD synthesis and degradation is important in the systemic plant response to developmental cues during the growth-phase transition.