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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., (© 2023. The Author(s).)

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 H 2 O 2 and O 2 - , which were reduced in the complementary line. Under short-day conditions, the nadk2 mutant accumulated more H 2 O 2 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., (© 2022. The Author(s) under exclusive licence to The Botanical Society of Japan.)

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 MgSO 4 , 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 MgSO 4 supplementation.

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 Ca 2+ -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., Competing Interests: Competing interestsThe authors declare that here are no conflicts of interest., (Copyright © 2020 The Authors; exclusive licensee Bio-protocol LLC.)

Over the last decade, combination of drugs in all stages of pharmaceutical development has accelerated availability of promising new therapies for difficult to treat diseases. Safety assessment of combined drugs to be tested in humans can occur at a critical path prior to proceeding in clinical testing. A recent survey by The International Consortium for Innovation and Quality in Pharmaceutical Development (IQ DruSafe) summarized member companies' approaches to combination safety strategies. In addition, feedback from Health Authorities (HAs) support a case-by-case scientific approach in assessing combination products' safety in accordance with the International Council on Harmonization (ICH) guidelines. Here, we present Pfizer's drug combination safety approach for various therapeutic areas (TA) including inflammation and immunology, metabolic, and anti-cancer products. There is no one-size-fits-all approach; rather, our main considerations include: strength of the existing clinical safety data for the individual compounds, common target organs, the potential for a synergistic effect, potential drug-drug interaction, routes of administration of each product and disease indications. No formal toxicity studies are considered necessary for anti-cancer drugs, while safety endpoints may be collected in preclinical pharmacology studies especially when the combined drugs present a novel mechanism. Combination safety studies when conducted for non-cancer indications can range from 2 to 13-weeks in duration, conducted usually in rodents, with dosages of individual molecules within clinical pharmacologic ranges. A case-by-case strategy guided by scientific rationale and in close collaboration with HAs remains the best approach to decide on the design and conduct of combination safety studies.

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 m 2 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 m 2 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 ( 134 Cs and 137 Cs). 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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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 CO 2 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.

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., Competing Interests: Competing interestsThe authors declare that there are no conflicts of interest., (Copyright © 2019 The Authors; exclusive licensee Bio-protocol LLC.)

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., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

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., (© The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Radiocesium ( 137 Cs) 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, 137 Cs 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 137 Cs 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 137 Cs 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 137 Cs 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 137 Cs translocation would cease considerably earlier than the cessation of K translocation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (© 2013 John Wiley & Sons Ltd.)

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., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.

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.

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 ((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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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., (© 2010 The Authors. The Plant Journal © 2010 Blackwell Publishing Ltd.)

Nicotinamide adenine dinucleotide (NAD) and its derivative nicotinamide adenine dinucleotide phosphate (NADP) are indispensable co-factors in broad-spectrum metabolic events for the maintenance of cellular homeostasis in all living organisms. In this study, the cellular expression levels of NAD biosynthesis genes in Arabidopsis were investigated. A very high expression of nicotinate/nicotinamide mononucleotide adenyltransferase (NMNAT) was observed in the differentiated stomatal guard cells of the leaf surface. Transcriptional analysis confirmed that several genes in the biosynthesis pathway were also highly expressed in stomatal guard cells. In fact, NAD and NADP metabolisms were investigated during stomatal movement. Importantly, the generation of phytohormone ABA-induced reactive oxygen species, which acts as a signal for stomatal closure, was accompanied by markedly decreased levels of NAD. The ABA-induced oxidative stress caused stomatal cell death in the nmnat mutant. Furthermore, stomata partially lost their ability to close leading to drought susceptibility. The stomata were less responsive to opening cues as well. These results indicate that NAD biosynthesis is involved in protecting guard cells from ABA-induced local oxidative stress via the regulation of NMNAT activity. In this study, it is demonstrated that NMNAT is essential for the maintenance of NAD homeostasis enabling sustainable stomatal movement.

The chloroplastic NAD kinase (NADK2) is reported to stimulate carbon and nitrogen assimilation in Arabidopsis (Arabidopsis thaliana), which is vulnerable to high light. Since rice (Oryza sativa) is a monocotyledonous plant that can adapt to high light, we studied the effects of NADK2 expression in rice by developing transgenic rice plants that constitutively expressed the Arabidopsis chloroplastic NADK gene (NK2 lines). NK2 lines showed enhanced activity of NADK and accumulation of the NADP(H) pool, while intermediates of NAD derivatives were unchanged. Comprehensive analysis of the primary metabolites in leaves using capillary electrophoresis mass spectrometry revealed elevated levels of amino acids and several sugar phosphates including ribose-1,5-bisphosphate, but no significant change in the levels of the other metabolites. Studies of chlorophyll fluorescence and gas change analyses demonstrated greater electron transport and CO2 assimilation rates in NK2 lines, compared to those in the control. Analysis of oxidative stress response indicated enhanced tolerance to oxidative stress in these transformants. The results suggest that NADP content plays a critical role in determining the photosynthetic electron transport rate in rice and that its enhancement leads to stimulation of photosynthesis metabolism and tolerance of oxidative damages.

Nicotinamide nucleotides (NAD and NADP) are important cofactors in many metabolic processes in living organisms. In this study, we analyzed transgenic Arabidopsis (Arabidopsis thaliana) plants that overexpress NAD kinase2 (NADK2), an enzyme that catalyzes the synthesis of NADP from NAD in chloroplasts, to investigate the impacts of altering NADP level on plant metabolism. Metabolite profiling revealed that NADP(H) concentrations were proportional to NADK activity in NADK2 overexpressors and in the nadk2 mutant. Several metabolites associated with the Calvin cycle were also higher in the overexpressors, accompanied by an increase in overall Rubisco activity. Furthermore, enhanced NADP(H) production due to NADK2 overexpression increased nitrogen assimilation. Glutamine and glutamate concentrations, as well as some other amino acids, were higher in the overexpressors. These results indicate that overexpression of NADK2 either directly or indirectly stimulates carbon and nitrogen assimilation in Arabidopsis under restricted conditions. Importantly, since neither up-regulation nor down-regulation of NADK2 activity affected the sum amount of NAD and NADP or the redox state, the absolute level of NADP and/or the NADP/NAD ratio likely plays a key role in regulating plant metabolism.

Background: Pyridine nucleotides are essential for electron transport and serve as co-factors in multiple metabolic processes in all organisms. Each nucleotide has a particular role in metabolism. For instance, the NAD/NADP ratio is believed to be responsible for sustaining the functional status of plant cells. However, since enzymes involved in the synthesis and degradation of NAD and NADP have not been fully identified, the physiological functions of these co-enzymes in plant growth and development are largely unknown., Scope: This Botanical Briefing covers progress in the developmental and stress-related roles of genes associated with NAD biosynthesis in plants. Special attention will be given to assessments of physiological impacts through the modulation of NAD and NADP biosynthesis., Conclusions: The significance of NAD biosynthesis in plant development and NADP biosynthesis in plant stress tolerance is summarized in this Briefing. Further investigation of cells expressing a set of NAD biosynthetic genes would facilitate understanding of regulatory mechanisms by which plant cells maintain NAD homeostasis.

While mammals and fungi possess nicotinate/nicotinamide mononucleotide adenyltransferase (NMNAT) isoforms, Arabidopsis thaliana only contains a single NMNAT gene, AtNMNAT (At5g55810). We analyzed the enzymatic activity of the AtNMNAT-encoded protein to determine the role of AtNMNAT in plant development. AtNMNAT catalyzed the synthesis of nicotinate adenine dinucleotide (NaAD) from nicotinate mononucleotide (NaMN) in the Preiss-Handler-dependent pathway, and of nicotinamide adenine dinucleotide (NAD) from nicotiamide mononucleotide (NMN) in the Preiss-Handler-independent pathway. Prominent AtNMNAT expression was detected in the male gametophyte. Moreover, AtNMNAT expression was spatio-temporally regulated during microspore development and pollen tube growth. Disruption of the AtNMNAT gene (atnmnat mutant) was characterized by a decrease in NAD content in pollen. Cytological examinations revealed that the atnmnat mutant was gametophytically impaired in in vivo and in vitro pollen tube growth. Our results suggest that metabolic fulfillment via the NAD pathway is indispensable for normal pollen growth and subsequent normal seed production.

Photosynthetic parameters of the nadk2 mutant of Arabidopsis thaliana, which is defective in chloroplast NAD kinase, were investigated. In this plant, the effective efficiency of photosynthetic electron transport (PhiII) and the quantum yield of open reaction centers of photosystem II (Fv'/Fm') were decreased. Furthermore, an increase in non-photochemical quenching attributed to energy dissipation from the xanthophyll cycle was observed. The mutant showed an aberrant de-epoxidation state of xanthophyll cycle carotenoids and had a high level of zeaxanthin even under low light conditions. These results indicate that chloroplast NAD kinase, catalyzing phosphorylation of NAD, is essential for the proper photosynthetic machinery of PSII and the xanthophyll cycle.

The Antirrhinum majus transposon Tam3 undergoes low temperature-dependent transposition (LTDT). Growth at 15 degrees C permits transposition, whereas growth at 25 degrees C strongly suppresses it. The degree of Tam3 DNA methylation is altered somatically and positively correlated with growth temperature, an exceptional epigenetic system in plants. Using a Tam3-inactive line, we show that methylation change depends on Tam3 activity. Random binding site selection analysis and electrophoretic mobility shift assays revealed that the Tam3 transposase (TPase) binds to the major repeat in the subterminal regions of Tam3, the site showing the biggest temperature-dependent change in methylation state. Methylcytosines in the motif impair the binding ability of the TPase. Proteins in a nuclear extract from plants grown at 15 degrees C but not 25 degrees C bind to this motif in Tam3. The decrease in Tam3 DNA methylation at low temperature also requires cell division. Thus, TPase binding to Tam3 occurs only during growth at low temperature and immediately after DNA replication, resulting in a Tam3-specific decrease in methylation of transposon DNA. Consequently, the Tam3 methylation level in LTDT is regulated by Tam3 activity, which is dependent on the ability of its TPase to bind DNA and affected by growth temperature. Thus, the methylation/demethylation of Tam3 is the consequence, not the cause, of LTDT.

It has been proposed that DNA methylation plays an important role in the inactivation of transposons. This view stems from a comparison of the degree of methylation of transposons in the active and inactive state. However, direct evidence for the degree of methylation required for the suppression of transposition has not been reported. Transposon Tam3 in Antirrhinum majus undergoes somatic reversal of its transposition activity, which is tightly controlled by temperature: low temperature around 15 degrees C permits transposition, high temperatures around 25 degrees C strongly inhibits it. Our previous study had shown that the methylation state of the Tam3 end regions is negatively correlated with the Tam3 transposition frequency. The results of the present study reveal that the inactive state of Tam3 copies at high temperature is unlikely to be directly coupled to the methylation state. Treatment with methylation inhibitors (5-azacytidine or 5-azacytidine+ethionine) does not affect Tam3 excision frequency in calli derived from Antirrhinum hypocotyls. The results suggest that methylation is not essential for the suppression of Tam3 transposition at high temperature, but rather that some other mechanism(s) involved in the control of Tam3 transposition may be obscured by methylation.

The transposition frequency of Tam3 in Antirrhinum majus, unlike that of most other cut-and-paste-type transposons, is tightly controlled by temperature: Tam3 transposes rarely at 25 degrees C, but much more frequently at 15 degrees C. Here, we studied the mechanism of the low-temperature-dependent transposition (LTDT) of Tam3. Our results strongly suggest that LTDT is not likely to be due to either transcriptional regulation or posttranscriptional regulation of the Tam3 TPase gene. We found that temperature shift induced a remarkable change of the methylation state unique to Tam3 sequences in the genome: Higher temperature resulted in hypermethylation, whereas lower temperature resulted in reduced methylation. The methylation state was reversible within a single generation in response to a temperature shift. Although our data demonstrate a close link between LTDT and the methylation of Tam3, they also suggest that secondary factor(s) other than DNA methylation is involved in repression of Tam3 transposition.

We identified eight independent Tam3 copies residing in the same Antirrhinum majus genome. All the copies showed excision at 15 degrees C, but not at 25 degrees C. Under conditions promoting excision, each copy appeared to transpose in the leaves and flower lobes with a nearly constant frequency, whereas individual transposition abilities varied widely: the most active copy had an excision frequency more than 100-fold greater than that of the least active one. Despite the different transposition abilities, the structures of the eight Tam3 copies were almost identical. These results made it clear that the transpositional ability of Tam3 is regulated by chromosomal position, but they do not imply position-dependent transposase activity. The position effect of the Tam3 transposition was found to be correlated to the methylation state of the copy's end regions: DNA methylation in the Tam3 end regions tended to suppress the excision activity, and the degree of methylation was dependent on the chromosomal position. Our results also provide evidence of de novo methylation provoked by transposition of the endogenous element. We propose a mechanism of transpositional regulation of plant transposons that responds to the degree of methylation as determined by chromosomal position.