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.

Radiocesium contamination in living and dead foliar parts of Japanese cedar during 2011-2015.

Radiocesium (137Cs) activity concentrations, mainly derived from the Fukushima accident of March 2011, were measured in green foliar parts without separation by age (bulk green foliar parts; GL) and litterfall (LF) of Japanese cedar (Cryptomeria japonica) from 2011 to 2015. In all samples, 137Cs concentrations decreased exponentially over time, but were always higher in LF (7.36-0.58 Bq g-DW-1) than in GL (2.10-0.06 Bq g-DW-1). The difference in the decreasing rate between GL and LF would reflect a difference in the dominant factor of the decrease between living and dead tissues (i.e., internal translocation and weathering, respectively). Over this same timeframe, potassium (K) concentrations in both GL and LF experienced repetitive periodical changes within a certain range (0.38-3.0 mg g-DW-1 for LF and 2.08-4.77 mg g-DW-1 for GL, respectively). Thus, there was no specific correlation between 137Cs and K concentrations in LF and GL. However, analyses of the age classified green foliar parts (GL-S) and dead foliar parts still retained on trees (DL) could indicate another view. The annual changes in residual rates of both 137Cs and K concentrations in GL-S demonstrated very similar two-phase reductions (i.e., a faster reduction in each expansion year than in the following years) and an obvious linear correlation between each other. Radiocesium concentration in DL were always higher than in any part of GL-S sampled at the same timing, but K concentrations showed the reverse relation. It is probable that 137Cs is basically translocated from older parts to the developing parts (as long as the former are alive) via a seasonal nutritional flow of K; however, a part of 137Cs translocation would cease considerably earlier than the cessation of K translocation.

Disparate radiocesium leaching from two woody species by acceleration of litter decomposition using microbial inoculation.

Studies focusing on the migration of radionuclides in the forest floor have demonstrated that the ecological half-life of radiocesium on organic layer containing the debris of plant litter with various fungi and microorganisms is shorter than that in the deeper soil zone, suggesting that the litter decomposition affects radiocesium mobilization. Here, we showed the involvement of lignin, one of the major cell wall components of plant litter, in the fate of contaminated radiocesium during the process of fungal litter decomposition. In this study, litter decomposition of two different woody species, broadleaf deciduous Japanese cherry consisted of hardwood lignin and coniferous evergreen Japanese cedar with softwood lignin, were accelerated by in vitro fungal inoculation. In vitro inoculation exhibited 1.93- to 2.59-times faster decomposition than field experiment. Then, the cherry litter lost approximately 25% of initially contaminated radiocesium within 1 month of in vitro decomposition, whereas the cedar litter kept initial level at least for 6 month. The retention of radiocesium correlated with thioglycolate lignin content in cedar litter but not in cherry litter. Consequently, the behavior of radiocesium contaminated in litter fall may vary depending on the contamination pathway or the manner of nutrient mobilization at the stage of abscission between evergreen and deciduous trees.

Changes in radiocesium contamination from Fukushima in foliar parts of 10 common tree species in Japan between 2011 and 2013.

Yearly changes in radiocesium ((137)Cs) contamination, primarily due to the Fukushima accident of March 2011, were observed in the foliar parts of 10 common woody species in Japan (Chamaecyparis obtusa, Cedrus deodara, Pinus densiflora, Cryptomeria japonica, Phyllostachys pubescens, Cinnamomum camphora, Metasequoia glyptostroboides, Prunus × yedoensis, Acer buergerianum, and Aesculus hippocastanum). The samples were obtained from Abiko (approximately 200 km SSW of the Fukushima Dai-ichi Nuclear Power Plant) during each growing season between 2011 and 2013, and the foliar parts were examined based on their year of expansion and location in each trees. The radiocesium concentrations generally decreased with time; however, the concentrations and rates of decrease varied among species, age of foliar parts, and locations. The radiocesium concentrations in the 2012 current-year foliar parts were 29%-220% of those from 2011, while those from 2013 fell to between 14% and 42% of the 2011 values. The net decontamination in the foliage was higher in evergreen species than in deciduous species. The radiocesium concentrations in the upper foliar parts were higher than those in the lower parts particularly in C. japonica. In addition, the radiocesium concentrations were higher in the current-year foliar parts than in the 1-year-old foliar parts, particularly in 2013. Thus, the influence of the direct deposition of the fallout was reduced with time, and the translocation ability of radiocesium from old to new tissues became more influential. Similar to the behavior of potassium in trees, Cs redistribution probably occurred primarily due to internal nutrient translocation mechanisms.

A kinetic analysis of cadmium accumulation in a Cd hyper-accumulator fern, Athyrium yokoscense and tobacco plants.

Cadmium (Cd) accumulations in a Cd hyper-accumulator fern, Athyrium yokoscense (Ay), and tobacco, Nicotiana tabacum (Nt), were kinetically analysed using the positron-emitting tracer imaging system under two medium conditions (basal and no-nutrient). In Ay, maximumly 50% and 15% of the total Cd accumulated in the distal roots and the shoots under the basal condition, respectively. Interestingly, a portion of the Cd in the distal roots returned to the medium. In comparison with Ay, a little fewer Cd accumulations in the distal roots and clearly higher Cd migration to the shoots were observed in Nt under the basal condition (maximumly 40% and 70% of the total Cd, respectively). The no-nutrient condition down-regulated the Cd migration in both species, although the regulation was highly stricter in Ay than in Nt (almost no migration in Ay and around 20% migration in Nt). In addition, the present work enabled to estimate physical and physiological Cd accumulation capacities in the distal roots, and demonstrated condition-dependent changes especially in Ay. These results clearly suggested occurrences of species-/condition-specific regulations in each observed parts. It is probable that integration of these properties govern the specific Cd tolerance/accumulation in Ay and Nt.

A time dependent behavior of radiocesium from the Fukushima-fallout in litterfalls of Japanese flowering cherry trees.

Radiocesium ((134)Cs + (137)Cs) concentrations, primarily derived from the Fukushima accident in March 2011, were measured in litterfalls and green leaves of Japanese flowering cherry trees (Prunus x yedoensis cv. Somei-Yoshino). The sampling was performed mainly during the defoliation season in 2011 and 2012 using traps to collect litterfalls before contact with the ground. The average radiocesium concentration in litterfalls in 2012 fell to one-third of that in 2011 (0.43 and 1.2 kBq kg-DW(-1), respectively). Interestingly, the concentrations in litterfalls collected in late autumn in both 2011 and 2012 (0.68 and 0.19 kBq kg-DW(-1), respectively) were significantly lower than those in litterfalls collected in the early autumn (1.7 and 1.1 kBq kg-DW(-1), respectively). In addition, the reductions in radiocesium concentrations in the litterfall were nearly synchronous with those in potassium concentrations (p ≤ 0.05). On the contrary, radiocesium concentrations in green leaves were also correlated with potassium concentrations; however, the slopes of the regression lines between the radiocesium and potassium concentrations were very similar in the 2011 litterfalls and the 2012 litterfalls, while the slopes were significantly different between these litterfalls and the green leaves. Consequently, the correlation between potassium and radiocesium was clear but independently observable in each of the litterfalls and the green leaves. It is possible that the reduction in radiocesium concentration occurred as a part of physiological demand, a translocation of potassium from the leaves to the body/twigs.

Real-time N2O gas detection system for agricultural production using a 4.6-µm-band laser source based on a periodically poled LiNbO3 ridge waveguide.

This article describes a gas monitoring system for detecting nitrous oxide (N2O) gas using a compact mid-infrared laser source based on difference-frequency generation in a quasi-phase-matched LiNbO3 waveguide. We obtained a stable output power of 0.62 mW from a 4.6-µm-band continuous-wave laser source operating at room temperature. This laser source enabled us to detect atmospheric N2O gas at a concentration as low as 35 parts per billion. Using this laser source, we constructed a new real-time in-situ monitoring system for detecting N2O gas emitted from potted plants. A few weeks of monitoring with the developed detection system revealed a strong relationship between nitrogen fertilization and N2O emission. This system is promising for the in-situ long-term monitoring of N2O in agricultural production, and it is also applicable to the detection of other greenhouse gases.

NAD⁺ accumulation as a metabolic off switch for orthodox pollen.

Terrestrial plant pollen is classified into two categories based on its metabolic status: pollen with low-metabolism are termed "orthodox" and pollen with high-metabolism are termed "recalcitrant." Nicotinamide adenine dinucleotide (NAD) is crucial for a number of metabolisms in all extant organisms. It has recently been shown that NAD homeostasis plays an important role in a broad range of developmental processes and responses to environment. Recently, a reverse genetic approach shed light on the significance of NAD biosynthesis on pollen fate. In orthodox Arabidopsis pollen, NAD(+) that was accumulated in excess at dispersal dramatically decreased on rehydration. The lack of a key gene that is involved in NAD biosynthesis compromised the excess accumulation. Moreover, absence of the excess accumulation phenocopied the so-called recalcitrant pollen, as demonstrated by the germination inside anthers and the loss of desiccation tolerance. Upon rehydration, NAD(+)-consuming inhibitors impaired tube germination. Taken together, our results suggest that accumulation of NAD(+) functions as a physiochemical molecular switch for suspended metabolism and that the decrease of NAD(+) plays a very important role during transitions in metabolic states. Shifting of the redox state to an oxidizing environment may efficiently control the comprehensive metabolic network underlying the onset of pollen germination.

NAD+ accumulation during pollen maturation in Arabidopsis regulating onset of germination.

Although the nicotinamide nucleotides NAD(H) and NADP(H) are essential for various metabolic reactions that play major roles in maintenance of cellular homeostasis, the significance of NAD biosynthesis is not well understood. Here, we investigated the dynamics of pollen nicotinamide nucleotides in response to imbibition, a representative germination cue. Metabolic analysis with capillary electrophoresis electrospray ionization mass spectrometry revealed that excess amount of NAD+ is accumulated in freshly harvested dry pollen, whereas it dramatically decreased immediately after contact with water. Importantly, excess of NAD+ impaired pollen tube growth. Moreover, NAD+ accumulation was retained after pollen was imbibed in the presence of NAD+-consuming reaction inhibitors and pollen germination was greatly retarded. Pollen deficient in the nicotinate/nicotinamide mononucleotide adenyltransferase (NMNAT) gene, encoding a key enzyme in NAD biosynthesis, and a lack of NAD+ accumulation in the gametophyte, showed precocious pollen tube germination inside the anther locule and vigorous tube growth under high-humidity conditions. Hence, the accumulation of excess NAD+ is not essential for pollen germination, but instead participates in regulating the timing of germination onset. These results indicate that NAD+ accumulation acts to negatively regulate germination and a decrease in NAD+ plays an important role in metabolic state transition.

Radiocesium contaminations of 20 wood species and the corresponding gamma-ray dose rates around the canopies at 5 months after the Fukushima nuclear power plant accident.

Radiocesium ((134)Cs + (137)Cs) deposition from the Fukushima nuclear power plant accident was measured in 20 woody plants (12 evergreen and 8 deciduous species) grown in Abiko (approximately 200 km SSW from the NPP). Leaves (needles) and twigs were sampled from each of three foliar positions (top, middle, and bottom) in the plant canopy in early August 2011. At the time, soils around the plants were also sampled, and gamma radiation dose rates were measured at each sampling position. The average radiocesium activity in the observed leaves of the evergreen species was 7.7 times that in the leaves of the deciduous species. Among the observed evergreen coniferous species, the activity in pre-fallout-expanded leaves was 2.4 times that in the post-fallout-expanded leaves. Notably, a distinct variation in the activity among the evergreen coniferous species could be observed for the post-fallout-expanded leaves but not for the pre-fallout-expanded leaves. Although these differences depend on whether the leaves had expanded at the time of the fallout, it is probable that a considerable amount of radiocesium was translocated to newly developed leaves at a species-specific rate. In addition, it was demonstrated that dose rates around woody plants were not consistent with the prevailing prediction that general dose rates correspondingly decrease with monitoring height from the ground. Thus, the dose rates in the top foliar layer of the deciduous species decreased more than predicted, whereas those in the top foliar layer of the coniferous species did not decrease. This may be due to differences in the balance between the attenuation resulting from a shielding effect of the plant bodies and the higher radiocesium accumulation in the leaves.

Temperature controls nuclear import of Tam3 transposase in Antirrhinum.

It has been proposed that environmental stimuli can activate transposable elements (TEs), whereas few substantial mechanisms have been shown so far. The class-II element Tam3 from Antirrhinum majus exhibits a unique property of low-temperature-dependent transposition (LTDT). LTDT has proved invaluable in developing the gene isolation technologies that have underpinned much of modern plant developmental biology. Here, we reveal that LTDT involves differential subcellular localization of the Tam3 transposase (TPase) in cells grown at low (15°C) and high (25°C) temperatures. The mechanism is associated with the nuclear import of Tam3 TPase in Antirrhinum cells. At high temperature, the nuclear import of Tam3 TPase is severely restricted in Antirrhinum cells, whereas at low temperature, the nuclear localization of Tam3 TPase is observed in about 20% of the cells. However, in tobacco BY-2 and Allium cepa (onion) cells, Tam3 TPase is transported into most nuclei. In addition to three nuclear localization signals (NLSs), the Tam3 TPase is equipped with a nuclear localization inhibitory domain (NLID), which functions to abolish nuclear import of the TPase at high temperature in Antirrhinum. NLID in Tam3 TPase is considered to interact with Antirrhinum-specific factor(s). The host-specific regulation of the nuclear localization of transposase represents a new repertoire controlling class-II TEs.