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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.

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.)

Maize root mitochondrial have been subfractionated after osmotic rupture. A calcium-calmodulin-dependent NAD-kinase activity has been shown to be present in both inner and outer membrane fractions. Cytochrome c-reductase activities are also associated with outer and inner membrane fractions but whereas the former is entirely insensitive to 50 μmol·1 antimycin A the latter is reduced by 60% in its presence. This residual antimycin A-insensitive cytochrome c-reductase activity cosediments with the major portion of NAD-kinase activity and equilibrates in sucrose gradients at densities around 1.146 g·cm. Rate zonal centrifugation with renografin allows an excellent separation of both cytochrome c-reductase and NAD-kinase activities. We have no evidence for allocating NAD-kinase activity to endo- or plasma membranes. [ABSTRACT FROM AUTHOR]

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NAD kinase was purified to homogeneity from Escherichia coli MG1655. The enzyme was a hexamer consisting of 30 kDa subunits and utilized ATP or other nucleoside triphosphates as phosphoryl donors for the phosphorylation of NAD, most efficiently at pH 7.5 and 60 °C. The enzyme could not use inorganic polyphosphates as phosphoryl donors and was designated as ATP–NAD kinase. The N-terminal amino-acid sequence of the purified enzyme was encoded by yfjB, which had been deposited as a gene of unknown function in the E. coli whole genomic DNA sequence database. yfjB was cloned and expressed in E. coli BL21(DE3)pLysS. The purified product (YfjB) showed NAD kinase activity, and was identical to ATP–NAD kinase purified from E. coli MG1655 in molecular structure and other enzymatic properties. The deduced amino-acid sequence of YfjB exhibited homology with that of Mycobacterium tuberculosis inorganic polyphosphate/ATP–NAD kinase [Kawai, S., Mori, S., Mukai, T., Suzuki, S., Hashimoto, W., Takeshi, Y. & Murata, K. (2000) Biochem. Biophys. Res. Commun. 276, 57–63], and those of many hypothetical proteins for which functions have not yet been revealed. The YfjB homologues were considered to be NAD kinases and alignment of their sequences revealed highly conserved regions, XXX-XGGDG-XL and DGXXX-TPTGSTAY, where X represents a hydrophobic amino-acid residue. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is a wide-spread gastrointestinal cancer that is associated with augmented morbidity and mortality, and we do not yet have a deep understanding of its epidemiology and carcinogenicity. The transcriptome can reveal the complexity and heterogeneity of tumors and uncover new biomarkers or treatment options. In this study, we identified messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), round RNAs (circRNAs), and microRNAs (miRNAs) using whole-transcriptome sequencing and generated competing endogenous RNA (ceRNA) modulatory axes. We conducted whole transcriptome sequencing on 10 CRC and para-cancer (CRCP) samples and discovered 2465 differentially expressed (DE) mRNAs (DEmRNAs), 77 DE miRNAs (DEmiRNAs). 2852 DE lncRNAs (DElncRNAs) and 1477 DE circRNAs (DEcircRNAs). In addition, utilizing co-DE analysis, we generated the ceRNA axis. Subsequently, we employed the ceRNA axis to identify essential genes and corresponding associations with lncRNAs, circRNAs, and miRNAs in CRC. ceRNA regulatory network including mRNA-miRNA-lncRNA and mRNA-miRNA-circRNA. These modulatory axes potentially modulate the positive regulation of smooth muscle contraction, melanosome, plasma membrane, integral plasma membrane component and so on. Finally, the results of RNA sequencing (RNA-SEQ) were combined with the TCGA and GEO databases, and the DEGs strongly correlated with the TCGA-COAD overall survival (OS) as estimated by univariate cox and logarithmic rank analyses were cross-analyzed, and the co-upregulated DEGs were screened. Among the many DEs, KPNA2 was chosen for additional analysis. Using invitro experimentations, western blot, CCK8, EdU and other experiments were performed to verify the results. We found siRNA-based KPNA2 depletion reduces bladder cancer cells' viability, migratory, and proliferative activities, which showed that the DEmRNA profiles were comparable to the sequencing information, confirming that the sequencing data were very reliable. These evidences highlight the ceRNA regulatory mechanisms in CRC and will aid future research into the molecular mechanisms behind colorectal cancer prevention and treatment. [ABSTRACT FROM AUTHOR]

NAD kinase was purified from pigeon liver by an improved procedure which included chromatography on phosphocellulose. The resultant preparation was homogeneous as judged by gel electrophoresis, but electrofocusing gave indications of heterogeneity. The enzyme appeared to be of molecular weight 270000, and to consist of subunits of molecular weight 34000; it may therefore be an octomer. Kinetic studies over a wide range of substrate concentrations revealed departures from Michaelis­Menten behaviour with the substrate NAD+; these were interpreted tentatively in terms of negative homotropic interactions between identical binding sites, since thermal and chemical inactivation studies revealed no evidence for more than one type of catalytic site. The significance of the kinetics and of the type of inhibition produced by NADPH is discussed in terms of the regulation of NAD kinase activity in vivo. [ABSTRACT FROM AUTHOR]

The remediation of copper and nickel-afflicted sites is challenged by the different physiological effects imposed by each metal on a given plant system. Pinus banksiana is resilient against copper and nickel, providing an opportunity to build a valuable resource to investigate the responding gene expression toward each metal. The objectives of this study were to (1) extend the analysis of the Pinus banksiana transcriptome exposed to nickel and copper, (2) assess the differential gene expression in nickel-resistant compared to copper-resistant genotypes, and (3) identify mechanisms specific to each metal. The Illumina platform was used to sequence RNA that was extracted from seedlings treated with each of the metals. There were 449 differentially expressed genes (DEGs) between copper-resistant genotypes (RGs) and nickel-resistant genotypes (RGs) at a high stringency cut-off, indicating a distinct pattern of gene expression toward each metal. For biological processes, 19.8% of DEGs were associated with the DNA metabolic process, followed by the response to stress (13.15%) and the response to chemicals (8.59%). For metabolic function, 27.9% of DEGs were associated with nuclease activity, followed by nucleotide binding (27.64%) and kinase activity (10.16%). Overall, 21.49% of DEGs were localized to the plasma membrane, followed by the cytosol (16.26%) and chloroplast (12.43%). Annotation of the top upregulated genes in copper RG compared to nickel RG identified genes and mechanisms that were specific to copper and not to nickel. NtPDR, AtHIPP10, and YSL1 were identified as genes associated with copper resistance. Various genes related to cell wall metabolism were identified, and they included genes encoding for HCT, CslE6, MPG, and polygalacturonase. Annotation of the top downregulated genes in copper RG compared to nickel RG revealed genes and mechanisms that were specific to nickel and not copper. Various regulatory and signaling-related genes associated with the stress response were identified. They included UGT, TIFY, ACC, dirigent protein, peroxidase, and glyoxyalase I. Additional research is needed to determine the specific functions of signaling and stress response mechanisms in nickel-resistant plants. [ABSTRACT FROM AUTHOR]

The present study explored the potential role of cold-regulated plasma membrane protein COR413PM1 isolated from Saussurea involucrata (Matsum. & Koidz)(SikCOR413PM1), in enhancing cotton (Gossypium hirsutum) tolerance to cold and drought stresses through transgenic methods. Under cold and drought stresses, the survival rate and the fresh and dry weights of the SikCOR413PM1-overexpressing lines were higher than those of the wild-type plants, and the degree of leaf withering was much lower. Besides, overexpressing SikCOR413PM1 overexpression increased the relative water content, reduced malondialdehyde content and relative conductivity, and elevated proline and soluble sugar levels in cotton seedlings. These findings suggest that SikCOR413PM1 minimizes cell membrane damage and boosts plant stability under challenging conditions. Additionally, overexpression of this gene upregulated antioxidant enzyme-related genes in cotton seedlings, resulting in enhanced antioxidant enzyme activity, lowered peroxide content, and reduced oxidative stress. SikCOR413PM1 overexpression also modulated the expression of stress-related genes (GhDREB1A, GhDREB1B, GhDREB1C, GhERF2, GhNAC3, and GhRD22). In field trials, the transgenic cotton plants overexpressing SikCOR413PM1 displayed high yields and increased environmental tolerance. Our study thus demonstrates the role of SikCOR413PM1 in regulating stress-related genes, osmotic adjustment factors, and peroxide content while preserving cell membrane stability and improving cold and drought tolerance in cotton. [ABSTRACT FROM AUTHOR]

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. NADP+ is the final electron acceptor for linear electron transfer in photosynthesis. Here, the authors show that the NADP pool size is modulated by its interconversion with NAD via ΔpH regulation in response to varying light conditions. [ABSTRACT FROM AUTHOR]

NAD kinase catalyzes the phosphorylation of NAD+ to synthesize NADP+, whereas NADH kinase catalyzes conversion of NADH to NADPH. The mitochondrial protein Pos5 of Saccharomyces cerevisiae shows much higher NADH kinase than NAD kinase activity and is therefore referred to as NADH kinase. To clarify the structural determinant underlying the high NADH kinase activity of Pos5 and its selectivity for NADH over NAD+, we determined the tertiary structure of Pos5 complexed with NADH at a resolution of 2.0 Å. Detailed analysis, including a comparison of the tertiary structure of Pos5 with the structures of human and bacterial NAD kinases, revealed that Arg-293 of Pos5, corresponding to His-351 of human NAD kinase, confers a positive charge on the surface of NADH-binding site, whereas the corresponding His residue does not. Accordingly, conversion of the Arg-293 into a His residue reduced the ratio of NADH kinase activity to NAD kinase activity from 8.6 to 2.1. Conversely, simultaneous changes of Ala-330 and His-351 of human NAD kinase into Ser and Arg residues significantly increased the ratio of NADH kinase activity to NAD kinase activity from 0.043 to 1.39; human Ala-330 corresponds to Pos5 Ser-272, which interacts with the side chain of Arg-293. Arg-293 and Ser-272 were highly conserved in Pos5 homologs (putative NADH kinases), but not in putative NAD kinases. Thus, Arg-293 of Pos5 is a major determinant of NADH selectivity. Moreover, Ser-272 appears to assist Arg-293 in achieving the appropriate conformation. [ABSTRACT FROM AUTHOR]

The commercial and nutritional value of strawberry fruit is drastically reduced due to its susceptibility to decay fungal diseases. In this study, we employed 40 μM riboflavin to delay senescence and decay of strawberry fruit during storage at 4 °C for 12 d. The findings showed that the treatment decreased strawberries mass loss, respiration rate, ROS level, and MDA content and maintained soluble sugars level. Riboflavin treatment maintained high levels of ATP content and energy charge, and activated the activities of succinate dehydrogenase (SDH), cytochrome C oxidase (CCO), H+-ATPase, Ca+-ATPase, glucokinase (GK), fructokinase (FRK), glucose-6-phosphate dehydrogenase (G6PDH), and 6-phosphogluconate dehydrogenase (6-P-GDH) and up-regulated the expression of their encoding genes. In addition, riboflavin-mediated NAD kinase (NADK) activation accelerated the conversion of NAD+ to DADP+ and the accumulation of DADPH. The respiratory pathway switched from the glycolytic (EMP)-tricarboxylic acid cycle (TCA) to the pentose phosphate pathway (PPP), reducing respiratory consumption. As a result, our findings suggest that riboflavin supplementation after harvest maintains a higher level of energy and delays senescence in strawberries. • Riboflavin treatment promoted strawberry respiration rate. • Riboflavin treatment maintained high levels of ATP content and energy charge. • Riboflavin treatment elevated NADPH contents and increased NAD kinase activity. • Enzyme activities involved in energy metabolism were enhanced by riboflavin treatment. [ABSTRACT FROM AUTHOR]

Background: With unique physiochemical environments in subcellular organelles, there has been growing interest in harnessing yeast organelles for bioproduct synthesis. Among these organelles, the yeast mitochondrion has been found to be an attractive compartment for production of terpenoids and branched-chain alcohols, which could be credited to the abundant supply of acetyl-CoA, ATP and cofactors. In this study we explored the mitochondrial potential for production of 3-hydroxypropionate (3-HP) and performed the cofactor engineering and flux control at the acetyl-CoA node to maximize 3-HP synthesis. Results: Metabolic modeling suggested that the mitochondrion serves as a more suitable compartment for 3-HP synthesis via the malonyl-CoA pathway than the cytosol, due to the opportunity to obtain a higher maximum yield and a lower oxygen consumption. With the malonyl-CoA reductase (MCR) targeted into the mitochondria, the 3-HP production increased to 0.27 g/L compared with 0.09 g/L with MCR expressed in the cytosol. With enhanced expression of dissected MCR enzymes, the titer reached to 4.42 g/L, comparable to the highest titer achieved in the cytosol so far. Then, the mitochondrial NADPH supply was optimized by overexpressing POS5 and IDP1, which resulted in an increase in the 3-HP titer to 5.11 g/L. Furthermore, with induced expression of an ACC1 mutant in the mitochondria, the final 3-HP production reached 6.16 g/L in shake flask fermentations. The constructed strain was then evaluated in fed-batch fermentations, and produced 71.09 g/L 3-HP with a productivity of 0.71 g/L/h and a yield on glucose of 0.23 g/g. Conclusions: In this study, the yeast mitochondrion is reported as an attractive compartment for 3-HP production. The final 3-HP titer of 71.09 g/L with a productivity of 0.71 g/L/h was achieved in fed-batch fermentations, representing the highest titer reported for Saccharomyces cerevisiae so far, that demonstrated the potential of recruiting the yeast mitochondria for further development of cell factories. [ABSTRACT FROM AUTHOR]

Polyphosphate kinases (PPKs) catalyze the reversible transfer of the γ-phosphate moiety of ATP (or of another nucleoside triphosphate) to a growing chain of polyphosphate (polyP). In this study, we describe that PPKs of various sources are additionally able to phosphorylate thiamine diphosphate (ThP2) to produce thiamine triphosphate (ThP3) and even thiamine tetraphosphate in vitro using polyP as phosphate donor. Furthermore, all tested PPK2s, but not PPK1s, were able to phosphorylate thiamine monophosphate (ThP1) to ThP2 and ThP3 although at low efficiency. The predicted masses and identities of the mono- and oligo-phosphorylated thiamine metabolites were identified by high-performance liquid chromatography tandem mass spectrometry. Moreover, the biological activity of ThP2, that was synthesized by phosphorylation of ThP1 with polyP and PPK, as a cofactor of ThP2-dependent enzymes (here transketolase TktA from Escherichia coli) was confirmed in a coupled enzyme assay. Our study shows that PPKs are promiscuous enzymes in vitro that could be involved in the formation of a variety of phosphorylated metabolites in vivo. [ABSTRACT FROM AUTHOR]

Boosting NAD+ levels are considered a promising means to promote healthy aging and ameliorate dysfunctional metabolism. The expression of CD38, the major NAD+-consuming enzyme, is downregulated during thermogenesis in both brown and white adipose tissues (BAT and WAT). Moreover, BAT activation and WAT "browning" were enhanced in Cd38−/− mice. In this study, the role of CD38 in the liver during thermogenesis was investigated, with the liver being the central organ controlling systemic energy metabolism. Wild-type mice and Cd38−/− mice were exposed to cold temperatures, and levels of metabolites and enzymes were measured in the livers and plasma. During cold exposure, CD38 expression was downregulated in the liver, as in BAT and WAT, with a concomitant increase in NAD(H) and a marked decrease in NADPH levels. Glucose-6-phosphate dehydrogenase and the malic enzyme, along with enzymes in the glycolytic pathway, were downregulated, which is in line with glucose-6-P being re-directed towards glucose release. In Cd38−/− mice, the cross-regulation between glycolysis and glucose release was lost, although this did not impair the glucose release from glycogen. Glycerol levels were decreased in the liver from Cd38−/− animals upon cold exposure, suggesting that glyceroneogenesis, as gluconeogenesis, was not properly activated in the absence of CD38. SIRT3 activity, regulating mitochondrial metabolism, was enhanced by cold exposure, whereas its activity was already high at a warm temperature in Cd38−/− mice and was not further increased by the cold. Notably, FGF21 and bile acid release was enhanced in the liver of Cd38−/− mice, which might contribute to enhanced BAT activation in Cd38−/− mice. These results demonstrate that CD38 inhibition can be suggested as a strategy to boost NAD+ and would not negatively affect hepatic functions during thermogenesis. [ABSTRACT FROM AUTHOR]

DNA methylation modification can regulate gene expression without changing the genome sequence, which helps organisms to rapidly adapt to new environments. However, few studies have been reported in non-model mammals. Giant panda (Ailuropoda melanoleuca) is a flagship species for global biodiversity conservation. Wildness and reintroduction of giant pandas are the important content of giant pandas' protection. However, it is unclear how wildness training affects the epigenetics of giant pandas, and we lack the means to assess the adaptive capacity of wildness training giant pandas. We comparatively analyzed genome-level methylation differences in captive giant pandas with and without wildness training to determine whether methylation modification played a role in the adaptive response of wildness training pandas. The whole genome DNA methylation sequencing results showed that genomic cytosine methylation ratio of all samples was 5.35%--5.49%, and the methylation ratio of the CpG site was the highest. Differential methylation analysis identified 544 differentially methylated genes (DMGs). The results of KEGG pathway enrichment of DMGs showed that VAV3, PLCG2, TEC and PTPRC participated in multiple immune-related pathways, and may participate in the immune response of wildness training giant pandas by regulating adaptive immune cells. A large number of DMGs enriched in GO terms may also be related to the regulation of immune activation during wildness training of giant pandas. Promoter differentially methylation analysis identified 1,199 genes with differential methylation at promoter regions. Genes with low methylation level at promoter regions and high expression such as, CCL5, P2Y13, GZMA, ANP32A, VWF, MYOZ1, NME7, MRPS31 and TPM1 were important in environmental adaptation for wildness training giant pandas. The methylation and expression patterns of these genes indicated that wildness training giant pandas have strong immunity, blood coagulation, athletic abilities and disease resistance. The adaptive response of giant pandas undergoing wildness training may be regulated by their negatively related promoter methylation. We are the first to describe the DNA methylation profile of giant panda blood tissue and our results indicated methylation modification is involved in the adaptation of captive giant pandas when undergoing wildness training. Our study also provided potential monitoring indicators for the successful reintroduction of valuable and threatened animals to the wild. [ABSTRACT FROM AUTHOR]

Arbuscular mycorrhizal fungi (AMF) are known to improve plant stress tolerance by regulating proline accumulation, and nitric oxide (NO) plays an important signaling role in proline metabolism. Environmental nitrogen (N) affects AMF colonization and its contribution to host plants resistance to stress conditions. However, the relationship between proline metabolism and NO in mycorrhizal rice and the effect of N application on symbiont proline metabolism under low temperature have not been established. Pot culture experiments with different temperature, N and exogenous NO donor treatments were conducted with non-mycorrhizal and mycorrhizal rice. The results showed that AMF enhanced rice proline accumulation under lowtemperature stress and decreased glutamate (Glu) and ornithine (Orn) concentrations significantly. In comparison with non-mycorrhizal rice, AMF colonization significantly decreased the Glu concentration, but had little effect on the Orn concentration under low-temperature stress, accompanied by increasing expression of OsP5CS2, OsOAT and OsProDH1. Exogenous application of NO increased proline concentration both under normal and low temperature, which exhibited a higher increase in mycorrhizal rice. NO also triggered the expression of key genes in the Glu and Orn pathways of proline synthesis as well as proline degradation. Higher N application decreased the AMF colonization, and AMF showed greater promotion of proline metabolism at low N levels under low temperature stress by regulating the Glu synthetic pathway. Meanwhile, AMF increased rice nitrate reductase (NR) and nitric oxide synthase (NOS) activities and then enhanced NO accumulation under low N levels. Consequently, it could be hypothesized that one of the mechanisms by which AMF improves plant resistance to low-temperature stress is the accumulation of proline via enhancement of the Glu and Orn synthetic pathways, with the involvement of the signaling molecule NO. However, the contribution of AMF to rice proline accumulation under low-temperature stress was attenuated by high N application. [ABSTRACT FROM AUTHOR]

Background: 7β-hydroxylated steroids (7β-OHSt) possess significant activities in anti-inflammatory and neuroprotection, and some of them have been widely used in clinics. However, the production of 7β-OHSt is still a challenge due to the lack of cheap 7β-hydroxy precursor and the difficulty in regio- and stereo-selectively hydroxylation at the inert C7 site of steroids in industry. The conversion of phytosterols by Mycolicibacterium species to the commercial precursor, androst-4-ene-3,17-dione (AD), is one of the basic ways to produce different steroids. This study presents a way to produce a basic 7β-hydroxy precursor, 7β-hydroxyandrost-4-ene-3,17-dione (7β-OH-AD) in Mycolicibacterium, for 7β-OHSt synthesis. Results: A mutant of P450-BM3, mP450-BM3, was mutated and engineered into an AD producing strain for the efficient production of 7β-OH-AD. The enzyme activity of mP450-BM3 was then increased by 1.38 times through protein engineering and the yield of 7β-OH-AD was increased from 34.24 mg L− 1 to 66.25 mg L− 1. To further enhance the performance of 7β-OH-AD producing strain, the regeneration of nicotinamide adenine dinucleotide phosphate (NADPH) for the activity of mP450-BM3-0 was optimized by introducing an NAD kinase (NADK) and a glucose-6-phosphate dehydrogenase (G6PDH). Finally, the engineered strain could produce 164.52 mg L− 1 7β-OH-AD in the cofactor recycling and regeneration system. Conclusions: This was the first report on the one-pot biosynthesis of 7β-OH-AD from the conversion of cheap phytosterols by an engineered microorganism, and the yield was significantly increased through the mutation of mP450-BM3 combined with overexpression of NADK and G6PDH. The present strategy may be developed as a basic industrial pathway for the commercial production of high value products from cheap raw materials. [ABSTRACT FROM AUTHOR]

Background: Frequent occurrence of extreme high temperature is a major threat to crop production. Increasing evidence demonstrates that long non-coding RNAs (lncRNAs) have important biological functions in the regulation of the response to heat stress. However, the regulatory mechanism of lncRNAs involved in heat response requires further exploration and the regulatory network remains poorly understood in maize. Results: In this research, high-throughput sequencing was adopted to systematically identify lncRNAs in maize inbred line CM1. In total, 53,249 lncRNAs (259 known lncRNAs and 52,990 novel lncRNAs) were detected, of which 993 lncRNAs showed significantly differential expression (DElncRNAs) under heat stress. By predicting the target genes, 953 common targets shared by cis- and trans-regulation of the DElncRNAs were identified, which exhibited differential expression between the control and the heat stress treatments. Functional annotation indicated that a number of important biological processes and pathways, including photosynthesis, metabolism, translation, stress response, hormone signal transduction, and spliceosome, were enriched for the common targets, suggesting that they play important roles in heat response. A lncRNA-mediated regulatory network was constructed to visualize the molecular response mechanism in response to heat stress, which represented the direct regulatory relationships of DElncRNAs, differentially expressed miRNAs, target genes, and functional annotations. Conclusions: This study lays a foundation for further elucidation of the regulatory mechanism for the response to heat stress in the maize inbred line CM1. The findings provide important information for identification of heat-responsive genes, which will be beneficial for the molecular breeding in the cultivation of heat-tolerant maize germplasm. [ABSTRACT FROM AUTHOR]

Cells of the yeast Saccharomyces cerevisiae contain three NAD kinases; namely, cytosolic Utrip, cytosolic Yefip, and mitochondrial Pos5p. Previously, the NADH kinase reaction catalyzed by Pos5p, rather than the NAD kinase reaction followed by the NADP[sup+]-dependent dehydrogenase reaction, had been regarded as a critical source of mitochondrial NADPH, which plays vital roles in various mitochondrial functions. This study demonstrates that the mitochondrial NADH kinase reaction is dispensable as a source of mitochondrial NADPH and emphasizes the importance of the NAD kinase reaction, followed by the mitochondrial NADP[sup+]-dependent dehydrogenase reaction. Of the potential dehydrogenases (malic enzyme, Maelp; isocitrate dehydrogenase, Idplp; and acetaldehyde dehydrogenases, Md4/5p), evidence is presented that acetaldehyde dehydrogenases, and in particular Ald4p, play a prominent role in generating mitochondrial NADPH in the absence of the NADH kinase reaction. The physiological significance of the mitochondrial NADH kinase reaction in the absence of Ald4p is also demonstrated. In addition, Pos5p is confirmed to have a considerably higher NADH kinase activity than NAD kinase activity. Taking these results together, it is proposed that there are two sources of mitochondrial NADPH in yeast: one is the mitochondrial Pos5pNADH kinase reaction and the other is the mitochondrial Pos5pNAD kinase reaction followed by the mitochondrial NADP[sup+]-dependent acetaldehyde dehydrogenase reaction. [ABSTRACT FROM AUTHOR]

Production of NADP and NADPH depends on activity of NAD and NADH kinases. Here we characterized all combinations of mutants in yeast NAD and NADH kinases to determine their physiological roles. We constructed a diploid strain heterozygous for disruption of POS5, encoding mitochondrial NADH kinase, UTR1, cytosolic NAD kinase, and YEF1, a UTR1-homologous gene we characterized as encoding a low specific activity cytosolic NAD kinase, pos5 utr1 is a synthetic lethal combination rescued by plasmid-borne copies of the POSS or UTR1 genes or by YEF1 driven by the ADH1 promoter. Respiratory-deficient and oxidative damage-sensitive defects in pos5 mutants were not made more deleterious by yell deletion, and a quantitative growth phenotype of pos5 and its arginine auxotrophy were repaired by plasmid-borne POS5 but not UTR1 or ADH1-driven YEF1. utr1 haploids have a slow growth phenotype on glucose not exacerbated by yef1 deletion but reversed by either plasmid-borne UTR1 or ADH1-driven YEF1. The defect in fermentative growth of utr1 mutants renders POSS but not POSS-dependent mitochondrial genome maintenance essential because rho-utr1 derivatives are viable. Purified Yef1 has similar nucleoside triphosphate specificity but substantially lower specific activity and less discrimination in favor of NAD versus NADH phosphorylation than Utr1. Low expression and low intrinsic NAD kinase activity of Yef1 and the lack of phenotype associated with yef1 suggest that Utr1 and Pos5 are responsible for essentially all NAD/NADH kinase activity in vivo. The data are compatible with a model in which there is no exchange of NADP, NADPH, or cytoplasmic NAD/NADH kinase between nucleocytoplasmic and mitochondrial compartments, but the cytoplasm is exposed to mitochondrial NAD/NADH kinase during the transit of the molecule. [ABSTRACT FROM AUTHOR]

In lowland rice ecosystems stagnant flooding or partial submergence has a significant negative impact on important yield attributing traits resulting in substantial grain yield reduction. Genetics of this stress is not yet studied intensively. Rashpanjor (IC 575321), a landrace from India, was identified and used as the tolerant donor for stagnant flooding and was crossed with high yielding variety Swarna to develop the RIL population for the present investigation. Yield and yield attributing traits of 180 F2:8 lines in rainfed non-stressed and stressed (stagnant flooding with 45 ± 5 cm standing water) conditions were recorded in the wet season of 2018 and stress susceptibility and tolerance indices of yield component traits were deduced. Homo-polymorphic high-quality SNPs between two parents derived from genotyping by sequencing were employed and 17 putative QTLs for plant height, shoot elongation, panicle number, grain weight, panicle length in control and stagnant flooding conditions were identified. Tolerance and susceptibility indexes for these traits were detected in chromosomes 1, 3, 4, 5, 6, 10, 11, and 12 with PVE ranging from 6.53 to 57.89%. Two major QTLs clusters were found for stress susceptibility index of grain and panicle weight on chromosome 1 and plant height in non-stress condition and stress tolerance index of elongation ability on chromosome 3. Putative functional genes present either in associated non-synonymous SNPs or inside the QTL regions were also predicted. Some of them were directly associated with ethylene biosynthesis and encoding auxin responsive factors for better adaptation under stagnant flooding and also coded for different transcription factors viz. NAC domain-binding protein, WRKY gene family, and MYB class known for ROS scavenging and production of metabolites to enhance tolerance to stagnant flooding. [ABSTRACT FROM AUTHOR]

Plant growth, development, and ultimately crop productivity are largely impacted by the interaction of plants with different abiotic and biotic factors throughout their life cycle. Perception of different abiotic stresses, such as salt, cold, drought, heat, and heavy metals, and interaction with beneficial and harmful biotic agents by plants lead to transient, sustained, or oscillatory changes of [calcium ion, Ca2+]cyt within the cell. Significant progress has been made in the decoding of Ca2+ signatures into downstream responses to modulate differential developmental and physiological responses in the whole plant. Ca2+ sensor proteins, mainly calmodulins (CaMs), calmodulin-like proteins (CMLs), and others, such as Ca2+-dependent protein kinases (CDPKs), calcineurin B-like proteins (CBLs), and calmodulin-binding transcription activators (CAMTAs) have played critical roles in coupling the specific stress stimulus with an appropriate response. This review summarizes the current understanding of the Ca2+ influx and efflux system in plant cells and various Ca2+ binding protein-mediated signal transduction pathways that are delicately orchestrated to mitigate abiotic and biotic stresses. The probable interactions of different components of Ca2+ sensor relays and Ca2+ sensor responders in response to various external stimuli have been described diagrammatically focusing on established pathways and latest developments. Present comprehensive insight into key components of the Ca2+ signaling toolkit in plants can provide an innovative framework for biotechnological manipulations toward crop improvability in near future. [ABSTRACT FROM AUTHOR]

Potassium (K+) deficiency severely threatens crop growth and productivity. Calcium (Ca2+) signaling and its sensors play a central role in the response to low-K+ stress. Calmodulin (CaM) is an important Ca2+ sensor. However, the mechanism by which Ca2+ signaling and CaM mediate the response of roots to low-K+ stress remains unclear. In this study, we found that the K+ concentration significantly decreased in both shoots and roots treated with Ca2+ channel blockers, a Ca2+ chelator, and CaM antagonists. Under low-K+ stress, reactive oxygen species (ROS) accumulated, and the activity of antioxidant enzymes, NAD kinase (NADK), and NADP phosphatase (NADPase) decreased. This indicates that antioxidant enzymes, NADK, and NADPase might be downstream target proteins in the Ca2+-CaM signaling pathway, which facilitates K+ uptake in plant roots by mediating ROS homeostasis under low-K+ stress. Moreover, the expression of NtCNGC3 , NtCNGC10 , K+ channel genes, and transporter genes was significantly downregulated in blocker-treated, chelator-treated, and antagonist-treated plant roots in the low K+ treatment, suggesting that the Ca2+-CaM signaling pathway may mediate K+ uptake by regulating the expression of these genes. Overall, this study shows that the Ca2+-CaM signaling pathway promotes K+ absorption by regulating ROS homeostasis and the expression of K+ uptake-related genes in plant roots under low-K+ stress. [ABSTRACT FROM AUTHOR]

Calmodulin and NAD kinase were extracted from serial developmental sections of the pea root apex. Highly purified samples of calmodulin were assayed by NAD-kinase activation, and whole-cell extracts were examined by two-dimensional polyacrylamide gel electrophoresis. Calmodulin was found to vary 17-fold in concentration over the apical 2 mm, being high in the region of the root cap and meristem, falling rapidly at the base of the meristem during early stages of rapid cell elongation. The rate of decline was different between stele and cortex. Except for a minor increase in concentration 2.5-5 mm from the apex, which coincides with the region of localised meristematic activity during initiation of lateral root primordia, the concentration of calmodulin remained at the lower level throughout the more basal sections of the apical 10 mm. In-vitro NAD-kinase activity was found to increase 17-fold per cell over the apical 30 mm, almost entirely as the result of an increase in calmodulin-dependent activity. Quantitative estimates of both calmodulin and NAD kinase were found to be highly dependent on extraction procedures. [ABSTRACT FROM AUTHOR]

Allium mongolicum Regel (A. mongolicum) is a perennial and xerophytic Liliaceous allium plant in high altitude desert steppe and desert areas. Feeding A. mongolicum greatly reduced unpleasant mutton flavor and improves meat quality of sheep. We analyzed epigenetic regulatory mechanisms of water extracts of A. mongolicum (WEA) on sheep muscle and adipose using RNA-Seq and whole-genome Bisulfite sequencing. Feeding WEA reduced differentially expressed genes and long non-coding RNAs (lncRNAs) between two tissues but increased differentially methylation regions (DMRs). LncRNA and DMR targets were both involved in ATP binding, ubiquitin, protein kinase binding, regulation of cell proliferation, and related signaling pathways, but not unsaturated fatty acids metabolism. Besides, tissue specific targets were involved in distinct functional annotations, e.g., Golgi membrane and endoplasmic reticulum for muscle lncRNA, oxidative phosphorylation metabolism for adipose lncRNA, dsRNA binding for muscle DMRs. Epigenetic regulatory networks were also discovered to discovered essential co-regulated modules, e.g., co-regulated insulin secretion module (PDPK1, ATP1A2, CACNA1S and CAMK2D) in adipose. The results indicated that WEA induced distinct epigenetic regulation on muscle and adipose to diminish transcriptome differences between tissues, which highlights biological functions of A. mongolicum, tissue similarity and specificity, as well as regulatory mechanism of mutton odor. [ABSTRACT FROM AUTHOR]

NADP+, the phosphorylated form of nicotinamide adenine dinucleotide (NAD), plays an essential role in many cellular processes. NAD kinase (NADK), which is conserved in all living organisms, catalyzes the phosphorylation of NAD+ to NADP+. However, the physiological role of phosphorylation of NAD+ to NADP+ in the cyanobacterium Synechocystis remains unclear. In this study, we report that slr0400 , an NADK-encoding gene in Synechocystis , functions as a growth repressor under light-activated heterotrophic growth conditions and light and dark cycle conditions in the presence of glucose. We show, via characterization of NAD(P)(H) content and enzyme activity, that NAD+ accumulation in slr0400 -deficient mutant results in the unsuppressed activity of glycolysis and tricarboxylic acid (TCA) cycle enzymes. In determining whether Slr0400 functions as a typical NADK, we found that constitutive expression of slr0400 in an Arabidopsis nadk2 -mutant background complements the pale-green phenotype. Moreover, to determine the physiological background behind the growth advantage of mutants lacking slr04000 , we investigated the photobleaching phenotype of slr0400 -deficient mutant under high-light conditions. Photosynthetic analysis found in the slr0400 -deficient mutant resulted from malfunctions in the Photosystem II (PSII) photosynthetic machinery. Overall, our results suggest that NADP(H)/NAD(H) maintenance by slr0400 plays a significant role in modulating glycolysis and the TCA cycle to repress the growth rate and maintain the photosynthetic capacity. [ABSTRACT FROM AUTHOR]

Plants can be simultaneously exposed to multiple stresses. The interplay of abiotic and biotic stresses may result in synergistic or antagonistic effects on plant development and health. Temporary drought stress can stimulate plant immunity; however, the molecular mechanism of drought-induced immunity is largely unknown. In this study, we demonstrate that cysteine protease RD21A is required for drought-induced immunity. Temporarily drought-treated wild-type Arabidopsis plants became more sensitive to the bacterial pathogen-associated molecular pattern flg22, triggering stomatal closure, which resulted in increased resistance to Pseudomonas syringae pv. tomato DC3000 (Pst -DC3000). Knocking out rd21a inhibited flg22-triggered stomatal closure and compromised the drought-induced immunity. Ubiquitin E3 ligase SINAT4 interacted with RD21A and promoted its degradation in vivo. The overexpression of SINAT4 also consistently compromised the drought-induced immunity to Pst -DC3000. A bacterial type III effector, AvrRxo1, interacted with both SINAT4 and RD21A, enhancing SINAT4 activity and promoting the degradation of RD21A in vivo. Therefore, RD21A could be a positive regulator of drought-induced immunity, which could be targeted by pathogen virulence effectors during pathogenesis. [ABSTRACT FROM AUTHOR]

Background: NAD kinases (NADKs) are the only known enzymes that directly phosphorylate NAD(H) to generate NADP(H) in different subcellular compartments. They participate in multiple life activities, such as modulating the NADP/NAD ratio, maintaining the intracellular redox balance and responding to environmental stresses. However, the functions of individual NADK in plants are still under investigation. Here, a rice NADK, namely, OsNADK1, was identified, and its functions in plant growth regulation and stress tolerance were analysed by employing a series of transgenic plant lines. Results: OsNADK1 is a cytosol-localized NADK in rice. It was expressed in all rice tissues examined, and its transcriptional expression could be stimulated by a number of environmental stress treatments. Compared with wild-type (WT) rice, the mutant plant osnadk1 in which OsNADK1 was knocked out was a dwarf at the heading stage and had decreased NADP(H)/NAD(H), ascorbic acid (ASA)/dehydroascorbate (DHA) and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios, which led to increased oxidation states in the rice cells and sensitivity to drought. Moreover, certain stress-related genes showed differential expression patterns in osnadk1 under both normal growth and drought-stress conditions compared with WT. Among these genes, OsDREB1B and several WRKY family transcription factors, e.g., OsWRKY21 and OsWRKY42, showed correlated co-expression patterns with OsNADK1 in osnadk1 and the plants overexpressing or underexpressing OsNADK1, implying roles for these transcription factors in OsNADK1-mediated processes. In addition, overexpression of OsNADK1 enhanced the drought tolerance of rice plants, whereas loss of function of the gene reduced the tolerance. Furthermore, the proline content was dramatically increased in the leaves of the OsNADK1-overexpressing lines under drought conditions. Conclusions: Altogether, the results suggest that an OsNADK1-mediated intracellular redox balance is involved in the tolerance of rice plants to drought. [ABSTRACT FROM AUTHOR]