Phosphoadenosine 5'-phosphate Research Articles

Crystal structure of activating sulfotransferase SgdX2 involved in biosynthesis of secondary metabolite sungeidine

Microorganisms synthesize a plethora of complex secondary metabolites, many of which are beneficial to human health, such as anticancer agents and antibiotics. Among these, the Sungeidines are a distinct class of secondary metabolites known for their bulky and intricate structures. They are produced by a specific biosynthetic gene cluster within the genome of the soil-dwelling actinomycete Micromonospora sp. MD118. A notable enzyme in the Sungeidine biosynthetic pathway is the activating sulfotransferase SgdX2. In this pathway, SgdX2 mediates a key sulfation step, after which the product undergoes spontaneous dehydration to yield a Sungeidine compound. To delineate the structural basis for SgdX2's substrate recognition and catalytic action, we have determined the crystal structure of SgdX2 in complex with its sulfate donor product, 3′-phosphoadenosine 5′-phosphate (PAP), at a resolution of 1.6 Å. Although SgdX2 presents a compact overall structure, its core elements are conserved among other activating sulfotransferases. Our structural analysis reveals a unique substrate-binding pocket that accommodates bulky, complex substrates, suggesting a specialized adaptation for Sungeidine synthesis. Moreover, we have constructed a substrate docking model that provides insights into the molecular interactions between SgdX2 and Sungeidine F, enhancing our understanding of the enzyme's specificity and catalytic mechanism. The model supports a general acid-base catalysis mechanism, akin to other sulfotransferases, and underscores the minor role of disordered regions in substrate recognition. This integrative study of crystallography and computational modeling advances our knowledge of microbial secondary metabolite biosynthesis and may facilitate the development of novel biotechnological applications.

Inhibition of RNA degradation integrates the metabolic signals induced by osmotic stress into the Arabidopsis circadian system.

The circadian clock system acts as an endogenous timing reference that coordinates many metabolic and physiological processes in plants. Previous studies have shown that the application of osmotic stress delays circadian rhythms via 3'-phospho-adenosine 5'-phosphate (PAP), a retrograde signalling metabolite that is produced in response to redox stress within organelles. PAP accumulation leads to the inhibition of exoribonucleases (XRNs), which are responsible for RNA degradation. Interestingly, we are now able to demonstrate that post-transcriptional processing is crucial for the circadian response to osmotic stress. Our data show that osmotic stress increases the stability of specific circadian RNAs, suggesting that RNA metabolism plays a vital role in circadian clock coordination during drought. Inactivation of XRN4 is sufficient to extend circadian rhythms as part of this response, with PRR7 and LWD1 identified as transcripts that are post-transcriptionally regulated to delay circadian progression.

Assimilatory sulfate reduction in the marine methanogen Methanothermococcus thermolithotrophicus

Methanothermococcus thermolithotrophicus is the only known methanogen that grows on sulfate as its sole sulfur source, uniquely uniting methanogenesis and sulfate reduction. Here we use physiological, biochemical and structural analyses to provide a snapshot of the complete sulfate reduction pathway of this methanogenic archaeon. We find that later steps in this pathway are catalysed by atypical enzymes. PAPS (3′-phosphoadenosine 5′-phosphosulfate) released by APS kinase is converted into sulfite and 3′-phosphoadenosine 5′-phosphate (PAP) by a PAPS reductase that is similar to the APS reductases of dissimilatory sulfate reduction. A non-canonical PAP phosphatase then hydrolyses PAP. Finally, the F420-dependent sulfite reductase converts sulfite to sulfide for cellular assimilation. While metagenomic and metatranscriptomic studies suggest that the sulfate reduction pathway is present in several methanogens, the sulfate assimilation pathway in M. thermolithotrophicus is distinct. We propose that this pathway was ‘mix-and-matched’ through the acquisition of assimilatory and dissimilatory enzymes from other microorganisms and then repurposed to fill a unique metabolic role.

Psychrobacter Phage Encoding an Antibiotics Resistance Gene Represents a Novel Caudoviral Family.

Psychrobacter is an important bacterial genus that is widespread in Antarctic and marine environments. However, to date, only two complete Psychrobacter phage sequences have been deposited in the NCBI database. Here, the novel Psychrobacter phage vB_PmaS_Y8A, infecting Psychrobacter HM08A, was isolated from sewage in the Qingdao area, China. The morphology of vB_PmaS_Y8A was characterized by transmission electron microscopy, revealing an icosahedral head and long tail. The genomic sequence of vB_PmaS_Y8A is linear, double-stranded DNA with a length of 40,226 bp and 44.1% G+C content, and encodes 69 putative open reading frames. Two auxiliary metabolic genes (AMGs) were identified, encoding phosphoadenosine phosphosulfate reductase and MarR protein. The first AMG uses thioredoxin as an electron donor for the reduction of phosphoadenosine phosphosulfate to phosphoadenosine phosphate. MarR regulates multiple antibiotic resistance mechanisms in Escherichia coli and is rarely found in viruses. No tRNA genes were identified and no lysogeny-related feature genes were detected. However, many similar open reading frames (ORFs) were found in the host genome, which may indicate that Y8A also has a lysogenic stage. Phylogenetic analysis based on the amino acid sequences of whole genomes and comparative genomic analysis indicate that vB_PmaS_Y8A contains a novel genomic architecture similar only to that of Psychrobacter phage pOW20-A, although at a low similarity. vB_PmaS_Y8A represents a new family-level virus cluster with 22 metagenomic assembled viral genomes, here named Minviridae. IMPORTANCE Although Psychrobacter is a well-known and important bacterial genus that is widespread in Antarctic and marine environments, genetic characterization of its phages is still rare. This study describes a novel Psychrobacter phage containing an uncharacterized antibiotic resistance gene and representing a new virus family, Minviridae. The characterization provided here will bolster current understanding of genomes, diversity, evolution, and phage-host interactions in Psychrobacter populations.

Coelenterazine sulfotransferase from Renilla muelleri.

The luciferin sulfokinase (coelenterazine sulfotransferase) of Renilla was previously reported to activate the storage form, luciferyl sulfate (coelenterazine sulfate) to luciferin (coelenterazine), the substrate for the luciferase bioluminescence reaction. The gene coding for the coelenterazine sulfotransferase has not been identified. Here we used a combined proteomic/transcriptomic approach to identify and clone the sulfotransferase cDNA. Multiple isoforms of coelenterazine sulfotransferase were identified from the anthozoan Renilla muelleri by intersecting its transcriptome with the LC-MS/MS derived peptide sequences of coelenterazine sulfotransferase purified from Renilla. Two of the isoforms were expressed in E. coli, purified, and partially characterized. The encoded enzymes display sulfotransferase activity that is comparable to that of the native sulfotransferase isolated from Renilla reniformis that was reported in 1970. The bioluminescent assay for sensitive detection of 3’-phosphoadenosine 5’-phosphate (PAP) using the recombinant sulfotransferase is demonstrated.

Transcriptomic and Metabolomic Response to High Light in the Charophyte Alga Klebsormidium nitens.

The characterization of the molecular mechanisms, such as high light irradiance resistance, that allowed plant terrestralization is a cornerstone in evolutionary studies since the conquest of land by plants played a pivotal role in life evolution on Earth. Viridiplantae or the green lineage is divided into two clades, Chlorophyta and Streptophyta, that in turn splits into Embryophyta or land plants and Charophyta. Charophyta are used in evolutionary studies on plant terrestralization since they are generally accepted as the extant algal species most closely related to current land plants. In this study, we have chosen the facultative terrestrial early charophyte alga Klebsormidium nitens to perform an integrative transcriptomic and metabolomic analysis under high light in order to unveil key mechanisms involved in the early steps of plants terrestralization. We found a fast chloroplast retrograde signaling possibly mediated by reactive oxygen species and the inositol polyphosphate 1-phosphatase (SAL1) and 3′-phosphoadenosine-5′-phosphate (PAP) pathways inducing gene expression and accumulation of specific metabolites. Systems used by both Chlorophyta and Embryophyta were activated such as the xanthophyll cycle with an accumulation of zeaxanthin and protein folding and repair mechanisms constituted by NADPH-dependent thioredoxin reductases, thioredoxin-disulfide reductases, and peroxiredoxins. Similarly, cyclic electron flow, specifically the pathway dependent on proton gradient regulation 5, was strongly activated under high light. We detected a simultaneous co-activation of the non-photochemical quenching mechanisms based on LHC-like stress related (LHCSR) protein and the photosystem II subunit S that are specific to Chlorophyta and Embryophyta, respectively. Exclusive Embryophyta systems for the synthesis, sensing, and response to the phytohormone auxin were also activated under high light in K. nitens leading to an increase in auxin content with the concomitant accumulation of amino acids such as tryptophan, histidine, and phenylalanine.

Differential genetic associations and expression of PAPST1/SLC35B2 in bipolar disorder and schizophrenia

Lithium's inhibitory effect on enzymes involved in sulfation process, such as inhibition of 3'(2')-phosphoadenosine 5'-phosphate (PAP) phosphatase, is a possible mechanism of its therapeutic effect for bipolar disorder (BD). 3'-Phosphoadenosine 5'-phosphosulfate (PAPS) is translocated from cytosol to Golgi lumen by PAPS transporter 1 (PAPST1/SLC35B2), where it acts as a sulfa donor. Since SLC35B2 was previously recognized as a molecule that facilitates the release of D-serine, a co-agonist of N-methyl-D-aspartate type glutamate receptor, altered function of SLC35B2 might be associated with the pathophysiology of BD and schizophrenia (SCZ). We performed genetic association analyses of the SLC35B2 gene using Japanese cohorts with 366 BD cases and 370 controls and 2012 SCZ cases and 2170 controls. We then investigated expression of SLC35B2 mRNA in postmortem brains by QPCR using a Caucasian cohort with 33 BD and 34 SCZ cases and 34 controls and by in situ hybridization using a Caucasian cohort with 37 SCZ and 29 controls. We found significant associations between three SNPs (rs575034, rs1875324, and rs3832441) and BD, and significantly reduced SLC35B2 mRNA expression in postmortem dorsolateral prefrontal cortex (DLPFC) of BD. Moreover, we observed normalized SLC35B2 mRNA expression in BD subgroups who were medicated with lithium. While there was a significant association of SLC35B2 with SCZ (SNP rs2233437), its expression was not changed in SCZ. These findings indicate that SLC35B2 might be differentially involved in the pathophysiology of BD and SCZ by influencing the sulfation process and/or glutamate system in the central nervous system.

Dissecting the Role of SAL1 in Metabolizing the Stress Signaling Molecule 3'-Phosphoadenosine 5'-Phosphate in Different Cell Compartments.

Plants possess the most highly compartmentalized eukaryotic cells. To coordinate their intracellular functions, plastids and the mitochondria are dependent on the flow of information to and from the nuclei, known as retrograde and anterograde signals. One mobile retrograde signaling molecule is the monophosphate 3′-phosphoadenosine 5′-phosphate (PAP), which is mainly produced from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) in the cytosol and regulates the expression of a set of nuclear genes that modulate plant growth in response to biotic and abiotic stresses. The adenosine bisphosphate phosphatase enzyme SAL1 dephosphorylates PAP to AMP in plastids and the mitochondria, but can also rescue sal1 Arabidopsis phenotypes (PAP accumulation, leaf morphology, growth, etc.) when expressed in the cytosol and the nucleus. To understand better the roles of the SAL1 protein in chloroplasts, the mitochondria, nuclei, and the cytosol, we have attempted to complement the sal1 mutant by specifically cargoing the transgenic SAL1 protein to these four cell compartments. Overexpression of SAL1 protein targeted to the nucleus or the mitochondria alone, or co-targeted to chloroplasts and the mitochondria, complemented most aspects of the sal1 phenotypes. Notably, targeting SAL1 to chloroplasts or the cytosol did not effectively rescue the sal1 phenotypes as these transgenic lines accumulated very low levels of SAL1 protein despite overexpressing SAL1 mRNA, suggesting a possibly lower stability of the SAL1 protein in these compartments. The diverse transgenic SAL1 lines exhibited a range of PAP levels. The latter needs to reach certain thresholds in the cell for its impacts on different processes such as leaf growth, regulation of rosette morphology, sulfate homeostasis, and glucosinolate biosynthesis. Collectively, these findings provide an initial platform for further dissection of the role of the SAL1–PAP pathway in different cellular processes under stress conditions.

Stomata: the holey grail of plant evolution

Stomata: the holey grail of plant evolution

Plastidial retrograde modulation of light and hormonal signaling: an odyssey.

The transition from an engulfed autonomous unicellular photosynthetic bacterium to a semiautonomous endosymbiont plastid was accompanied by the transfer of genetic material from the endosymbiont to the nuclear genome of the host, followed by the establishment of plastid-to-nucleus (retrograde) signaling. The retrograde coordinated activities of the two subcellular genomes ensure chloroplast biogenesis and function as the photosynthetic hub and sensing and signaling center that tailors growth-regulating and adaptive processes. This review specifically focuses on the current knowledge of selected stress-induced retrograde signals, genomes uncoupled 1 (GUN1), methylerythritol cyclodiphosphate (MEcPP), apocarotenoid and β-cyclocitral, and 3'-phosphoadenosine 5'-phosphate (PAP), which evolved to establish the photoautotrophic lifestyle and are instrumental in the integration of light and hormonal signaling networks to ultimately fashion adaptive responses in an ever-changing environment.

Evaluation of a conserved tryptophanyl residue in donor substrate binding and catalysis by a phenol sulfotransferase (SULT1A1)

Structural determinations of members of the sulfotransferase (SULT) family suggest a direct interaction between a conserved tryptophanyl side chain and bound 3′-phosphoadenosine-5′-phosphate (PAP). We have prepared and purified mutants of the bovine SULT1A1, a very conserved homolog to the human SULT1A1, in which tryptophanyl-53 was sequentially trimmed to tyrosine, leucine, and alanine. Differential scanning fluorimetry indicated structural stabilities of the mutant proteins comparable to the wild type SULT1A1; however, less thermal stabilizations by PAP plus pentachlorophenol were observed with the mutants, suggesting weakened ligand binding. Protein fluorescence of the wild type enzyme decreased 6.5% upon binding PAP, whereas no changes occurred with the mutant enzymes. This reveals that W53, or its positional counterpart, has been the source of emission intensity changes used in previous investigations of other SULTs. Fluorescence resonance energy transfer from excited tryptophans to bound 7-hydroxycoumarin, as induced by PAP, indicated weakened binding of ligands to the mutant SULTs. This was also encountered and quantified in initial rate kinetic analyses. Ablation of the PAPS adenine-to-W53 ring interaction, shown by the W53A mutant enzyme, resulted in a 6.4-fold increase in KPAPS and a 92% decrease in kcat/KPAPS. Measured KPAPS values reveal the W53 indole ring contribution to PAPS binding to be 1.1 kcal/mol (4.6 kJ/mol). These results verify the structurally-inferred role for the π−π stacking interaction between PAP(S) and the conserved tryptophanyl residue in SULT1A1 and other members of the SULT family.

Enzymatic Conversion of Cypridina Luciferyl Sulfate to Cypridina Luciferin with Coenzyme A as a Sulfate Acceptor in Cypridina (Vargula) hilgendorfii.

In the luminous ostracod Cypridina (presently Vargula) hilgendorfii, Cypridina luciferyl sulfate (3-enol sulfate of Cypridina luciferin) is converted to Cypridina luciferin by a sulfotransferase with 3'-phosphoadenosine-5'-phosphate (PAP) as a sulfate acceptor. The resultant Cypridina luciferin is used for the luciferase-luciferin reaction of Cypridina to emit blue light. The luminescence stimulation with major organic cofactors was examined using the crude extracts of Cypridina specimens, and we found that the addition of coenzyme A (CoA) to the crude extracts significantly stimulated luminescence intensity. Further, the light-emitting source in the crude extracts stimulated with CoA was identified as Cypridina luciferyl sulfate, and we demonstrated that CoA could act as a sulfate acceptor from Cypridina luciferyl sulfate. In addition, the sulfate group of Cypridina luciferyl sulfate was also transferred to adenosine 5'-monophosphate (5'-AMP) and adenosine 3'-monophosphate (3'-AMP) by a sulfotransferase. The sulfated products corresponding to CoA, 5'-AMP and 3'-AMP were identified using mass spectrometry. This is the first report that CoA can act as a sulfate acceptor in a sulfotransferase reaction.

Abstract 2151: Monitoring AMP Concentration,the universal product of diverse enzymatic reactions

Abstract Many enzymatic reactions use Adenosine triphosphate (ATP) as a substrate and produce adenosine monophosphate (AMP) as the common product of these reactions. AMP also functions as a modulator of many enzymes and a key cellular metabolite regulating energy homeostasis and signal transduction. For example, by activating AMP-Dependent protein kinase (AMPK), it maintains the energy charge ratio at optimal level during metabolic activities; and its concentration is altered during disregulated disease conditions. Thus, monitoring the activities of these enzymes is a primary goal for developing modulators for these enzymes. Here, we demonstrate the versatility of an enzyme coupled assay that quantifies the amount of AMP produced by any enzymatic reactions regardless of its substrates in a homogenous and high through-put formatted form. We successfully implemented it to enzyme reactions that use ATP as a substrate (aminoacyl tRNA synthetase and DNA ligase) by first depleting the remaining residual ATP substrate and converting AMP produced into ATP, the later can be quantified using luciferase/luciferin and generating light. We also tested this assay to measure direct activities of AMP generating enzymes that do not require ATP as substrate, including phosphodiesterases (cAMP) and E.coli DNA ligases (NAD+). In a further elaboration of the AMP-Glo platform, we coupled it to E.coli DNA ligase, enabling measurement of NAD+ and enzymes that use NAD+ like mono- and poly-ADP-ribosyltransferases. Sulfotransferases use 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as the universal sulfo-group donor and phosphoadenosine-5'-phosphate (PAP) is the universal product. PAP can be quantified by converting PAP to AMP by a Golgi-resident PAP-specific phosphatase, IMPAD1. By coupling IMPAD1 to the AMP-Glo system we can measure the activities of sulfotransferases. In summary, we have developed a very strong platform to monitor AMP concentrations that is applicable to diverse enzymatic reactions. Citation Format: Said A. Goueli, Kevin Hsiao, Subhanjan Mondal. Monitoring AMP Concentration,the universal product of diverse enzymatic reactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2151.

Secretory expression of the rat aryl sulfotransferases IV with improved catalytic efficiency by molecular engineering.

The rat aryl sulfotransferases IV (AST IV) has been used to catalyze 3'-phosphoadenosine-5'-phosphate (PAP) into the sulfuryl group donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) in biotechnological production of glycosaminoglycans. The performance of native AST IV is not satisfying due to the lower catalytic activity with substrate PAP. In the present study, we achieved secretory expression of the AST IV and improved its catalytic efficiency by molecular engineering. Fusion with signal peptides Cex, YebF and PelB allow for secretory expression of AST IV and the secreted AST IV yield reached 4.21 ± 0.23U/mL, 8.67 ± 0.34U/mL and 21.35 ± 0.87U/mL, respectively. Modification of PelB further increased the secretory expression by more than fourfold, to 89.67 ± 1.34U/mL. On this basis, molecular evolution of the predicted PAP-binding pocket gate loop was performed and a positive mutant L89S/E90L with higher activity was identified. Considering the importance of the sites Leu89 and Glu90, we performed site-saturation mutagenesis and found the mutant L89M/E90Q with much higher PAP affinity (K m= 0.46 ± 0.02mM) and catalytic efficiency (k cat/K m = 1816.32 ± 12.72/s/M). The secretory expression of the AST IV variant L89M/E90Q with higher catalytic efficiency should facilitate the studies on biosynthesis of sulfated polysaccharides.

Evolution of chloroplast retrograde signaling facilitates green plant adaptation to land

Chloroplast retrograde signaling networks are vital for chloroplast biogenesis, operation, and signaling, including excess light and drought stress signaling. To date, retrograde signaling has been considered in the context of land plant adaptation, but not regarding the origin and evolution of signaling cascades linking chloroplast function to stomatal regulation. We show that key elements of the chloroplast retrograde signaling process, the nucleotide phosphatase (SAL1) and 3'-phosphoadenosine-5'-phosphate (PAP) metabolism, evolved in streptophyte algae-the algal ancestors of land plants. We discover an early evolution of SAL1-PAP chloroplast retrograde signaling in stomatal regulation based on conserved gene and protein structure, function, and enzyme activity and transit peptides of SAL1s in species including flowering plants, the fern Ceratopteris richardii, and the moss Physcomitrella patens Moreover, we demonstrate that PAP regulates stomatal closure via secondary messengers and ion transport in guard cells of these diverse lineages. The origin of stomata facilitated gas exchange in the earliest land plants. Our findings suggest that the conquest of land by plants was enabled by rapid response to drought stress through the deployment of an ancestral SAL1-PAP signaling pathway, intersecting with the core abscisic acid signaling in stomatal guard cells.

Arabidopsis RCD1 coordinates chloroplast and mitochondrial functions through interaction with ANAC transcription factors.

Reactive oxygen species (ROS)-dependent signaling pathways from chloroplasts and mitochondria merge at the nuclear protein RADICAL-INDUCED CELL DEATH1 (RCD1). RCD1 interacts in vivo and suppresses the activity of the transcription factors ANAC013 and ANAC017, which mediate a ROS-related retrograde signal originating from mitochondrial complex III. Inactivation of RCD1 leads to increased expression of mitochondrial dysfunction stimulon (MDS) genes regulated by ANAC013 and ANAC017. Accumulating MDS gene products, including alternative oxidases (AOXs), affect redox status of the chloroplasts, leading to changes in chloroplast ROS processing and increased protection of photosynthetic apparatus. ROS alter the abundance, thiol redox state and oligomerization of the RCD1 protein in vivo, providing feedback control on its function. RCD1-dependent regulation is linked to chloroplast signaling by 3'-phosphoadenosine 5'-phosphate (PAP). Thus, RCD1 integrates organellar signaling from chloroplasts and mitochondria to establish transcriptional control over the metabolic processes in both organelles.

Loss of Arabidopsis Halotolerance 2-like (AHL), a 3'-phosphoadenosine-5'-phosphate phosphatase, suppresses insensitive response of Arabidopsis thaliana ring zinc finger 1 (atrzf1) mutant to abiotic stress.

Destruction of PAP phosphatase AHL suppresses atrzf1 phenotype in abiotic stress responses. AHL plays an intermediate role in the regulation of proline accumulation by PAP nucleotidase. Proline (Pro) metabolism is important for environmental responses, plant development, and growth. However, the role of Pro in abiotic stress process is unclear. Using atrzf1 (Arabidopsis thaliana ring zinc finger 1) mutant as a parental line for T-DNA tagging mutagenesis, we identified a suppressor mutant designated as proline content alterative 17 (pca17) that suppressed insensitivity of atrzf1 to abiotic stresses during early seedling growth. Pro content of pca17 was lower than that in both wild type (WT) and atrzf1 while complementary lines were less sensitive to abscisic acid (ABA) and abiotic stresses compared to WT. Thermal Asymmetric Interlaced (TAIL)-PCR of pca17 showed that T-DNA was inserted at site of At5g54390 (AHL for Arabidopsis Halotolerance 2-like) encoding 3'-phosphoadenosine-5'-phosphate (PAP) phosphatase. Under drought stress condition, products of sulfate metabolism such as PAP and adenosine monophosphate were significantly lower in pca17 than those in WT and atrzf1. Furthermore, pca17 showed significantly higher levels of several important drought parameters including malondialdehyde, ion leakage, and water loss than WT and atrzf1. Fluorescence signal of green fluorescent protein (GFP)-tagged AHL was quite strong in nuclei of the root and guard cells of transgenic seedlings. Additionally, AHL promoter-β-glucuronidase (GUS) construct revealed substantial gene expression in vasculature tissues and pollen. Collectively, these findings demonstrate that pca17 acts as a dominant suppressor mutant of atrzf1 in abiotic stress response by modulating proline and sulfate metabolism.

Chloroplast-to-Nucleus Signaling Regulates MicroRNA Biogenesis in Arabidopsis

As integral regulators in plant development and stress response, microRNAs (miRNAs) themselves need to be tightly regulated. Here, we show that tocopherols (vitamin E), lipid-soluble antioxidants synthesized from tyrosine in chloroplasts, positively regulate the biogenesis of miRNAs. Tocopherols are required for the accumulation of 3'-phosphoadenosine 5'-phosphate (PAP), a retrograde inhibitor of the nuclear exoribonucleases (XRN), which may protect primary miRNAs from being degraded and promote mature miRNA production. Such regulation is involved in heat-induced accumulation of miR398 and plant acquisition of heat tolerance. Our study reveals a chloroplast-to-nucleus signaling mechanism that favors miRNA biogenesis under heatand possibly other environmental perturbations.

The Arabidopsis SAL1-PAP Pathway: A Case Study for Integrating Chloroplast Retrograde, Light and Hormonal Signaling in Modulating Plant Growth and Development?

Plant growth and development are dependent on chloroplast development and function. Constitutive high level accumulation of a chloroplast stress signal, 3′-phosphoadenosine-5′-phosphate (PAP), confers drought tolerance to plants, but slow downs and alters plant growth and development. PAP, a by-product of sulfur metabolism, is maintained at very low levels by the SAL1 phosphatase during vegetative growth of Arabidopsis and accumulates in rosettes during drought and excess light. Eight independent forward genetic screens in Arabidopsis identified SAL1 as the regulator of multiple phenotypes related to stress responses, hormonal signaling and/or perception. In this perspective article, we collate all the sal1 phenotypes published in the past two decades, and distill the different pathways affected. Our meta-analysis of publicly available sal1 microarray data coupled to preliminary hormonal treatment and profiling results on sal1 indicate that homeostasis and responses to multiple hormones in sal1 are altered during rosette growth, suggesting a potential connection between SAL1-PAP stress retrograde pathway and hormonal signaling. We propose the SAL1-PAP pathway as a case study for integrating chloroplast retrograde signaling, light signaling and hormonal signaling in plant growth and morphogenesis.

Utility of AMP Detection System for Monitoring the Activities of Diverse Enzyme Reactions

Adenosine monophosphate (AMP) is a key cellular metabolite regulating energy homeostasis and signal transduction. AMP is also a product of various enzymatic reactions, many of which are disregulated during disease conditions. Thus, monitoring the activities of these enzymes is a primary goal for developing modulators for these enzymes. Here, we demonstrate the versatility of an enzyme coupled assay that quantifies the amount of AMP produced by any enzymatic reactions regardless of its substrates. We successfully implemented it to enzyme reactions that use ATP as a substrate (aminoacyl tRNA synthetase and DNA ligase) by an elaborate strategy of removing residual ATP and converting AMP produced into ATP so it can be detected using luciferase/luciferin and generating light. We also tested this assay to measure the activities of AMP generating enzymes that do not require ATP as substrate, including phosphodiesterases (cAMP) and E.coli DNA ligases (NAD+). In a further elaboration of the AMP‐Glo platform, we coupled it to E.coli DNA ligase, enabling measurement of NAD+ and enzymes that use NAD+ like mono‐ and poly‐ADP‐ribosyltransferases. Sulfotransferases use 3′‐Phosphoadenosine‐5′‐phosphosulfate (PAPS) as the universal sulfo‐group donor and Phosphoadenosine‐5′‐phosphate (PAP) is the universal product. PAP can be quantified by converting PAP to AMP by a Golgiresident PAP‐specific phosphatase, IMPAD1. By coupling IMPAD1 to the AMP‐Glo system we can measure the activities of sulfotransferases. Thus by utilizing the combinations of biochemical enzymatic conversion of various cellular metabolites to AMP, we were able to demonstrate the versatility of the AMP‐Glo assay.Support or Funding InformationThe research was supported by Promega Corp.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.