A Monochromatically Excitable Green-Red Dual-Fluorophore Fusion Incorporating a New Large Stokes Shift Fluorescent Protein.

Genetically encoded sensors enable quantitative imaging of analytes in live cells. Sensors are commonly constructed by combining ligand-binding domains with one or more sensitized fluorescent protein (FP) domains. Sensors based on a single FP can be susceptible to artifacts caused by changes in sensor levels or distribution in vivo. To develop intensiometric sensors with the capacity for ratiometric quantification, dual-FP Matryoshka sensors were generated by using a single cassette with a large Stokes shift (LSS) reference FP nested within the reporter FP (cpEGFP). Here, we present a genetically encoded calcium sensor that employs green apple (GA) Matryoshka technology by incorporating a newly designed red LSSmApple fluorophore. LSSmApple matures faster and provides an optimized excitation spectrum overlap with cpEGFP, allowing for monochromatic coexcitation with blue light. The LSS of LSSmApple results in improved emission spectrum separation from cpEGFP, thereby minimizing fluorophore bleed-through and facilitating imaging using standard dichroic and red FP (RFP) emission filters. We developed an image analysis pipeline for yeast (Saccharomyces cerevisiae) timelapse imaging that utilizes LSSmApple to segment and track cells for high-throughput quantitative analysis. In summary, we engineered a new FP, constructed a genetically encoded calcium indicator (GA-MatryoshCaMP6s), and performed calcium imaging in yeast as a demonstration.

Sensors for the quantification, localization and analysis of the dynamics of plant hormones.

Plant hormones play important roles in plant growth and development and physiology, and in acclimation to environmental changes. The hormone signaling networks are highly complex and interconnected. It is thus important to not only know where the hormones are produced, how they are transported and how and where they are perceived, but also to monitor their distribution quantitatively, ideally in a non-invasive manner. Here we summarize the diverse set of tools available for quantifying and visualizing hormone distribution and dynamics. We provide an overview over the tools that are currently available, including transcriptional reporters, degradation sensors, and luciferase and fluorescent sensors, and compare the tools and their suitability for different purposes.

Designs, applications, and limitations of genetically encoded fluorescent sensors to explore plant biology.

The understanding of signaling and metabolic processes in multicellular organisms requires knowledge of the spatial dynamics of small molecules and the activities of enzymes, transporters, and other proteins in vivo, as well as biophysical parameters inside cells and across tissues. The cellular distribution of receptors, ligands, and activation state must be integrated with information about the cellular distribution of metabolites in relation to metabolic fluxes and signaling dynamics in order to achieve the promise of in vivo biochemistry. Genetically encoded sensors are engineered fluorescent proteins that have been developed for a wide range of small molecules, such as ions and metabolites, or to report biophysical processes, such as transmembrane voltage or tension. First steps have been taken to monitor the activity of transporters in vivo. Advancements in imaging technologies and specimen handling and stimulation have enabled researchers in plant sciences to implement sensor technologies in intact plants. Here, we provide a brief history of the development of genetically encoded sensors and an overview of the types of sensors available for quantifying and visualizing ion and metabolite distribution and dynamics. We further discuss the pros and cons of specific sensor designs, imaging systems, and sample manipulations, provide advice on the choice of technology, and give an outlook into future developments.

The NAD Kinase Slr0400 Functions as a Growth Repressor in Synechocystis sp. PCC 6803.

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.

One of the NAD kinases, sll1415, is required for the glucose metabolism of Synechocystis sp. PCC 6803.

Pyridine nucleotides (NAD(P)(H)) are electron carriers that are the driving forces in various metabolic pathways. Phosphorylation of NAD(H) to NADP(H) is performed by the enzyme NAD kinase (NADK). Synechocystis sp. PCC 6803 harbors two genes (sll1415 and slr0400) that encode proteins with NADK homology. When genetic mutants for sll1415 and slr0400 (Δ1415 and Δ0400, respectively) were cultured under photoheterotrophic growth conditions only the Δ1415 cells showed a growth defect. In wild-type cells, the sll1415 transcript accumulated after the cells were transferred to photoheterotrophic conditions. Furthermore, NAD(P)(H) measurements demonstrated that a dynamic metabolic conversion was implemented during the adaptation from photoautotrophic to photoheterotrophic conditions. Electron microscopy observation and biochemistry quantification demonstrated the accumulation of glycogen in the Δ1415 cells under photoheterotrophic conditions at 96 h. Quantitative real-time reverse transcription PCR (qRT-PCR) demonstrated the accumulation of mRNAs that encoded glycogen biosynthesis-related enzymes in photoheterotrophic Δ1415 cells. At 96 h, enzyme activity measurement in the photoheterotrophic Δ1415 cells demonstrated that the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were decreased, but the activities of glucose dehydrogenase were increased. Furthermore, metabolomics analysis demonstrated that the Δ1415 cells showed increased glucose-6-phosphate and 6-phosphogluconate content at 96 h. Therefore, sll1415 has a significant function in the oxidative pentose phosphate (OPP) pathway for catabolism of glucose under photoheterotrophic conditions. Additionally, it is presumed that the slr0400 had a different role in glucose catabolism during growth. These results suggest that the two Synechocystis sp. PCC 6803 NADKs (Sll1415 and Slr0400) have distinct functions in photoheterotrophic cyanobacterial metabolism.

Heterologous expression of mtf and mtc genes of Pseudanabaena foetida var. intermedia is sufficient to produce 2-methylisoborneol in Escherichia coli.

Microbial volatile metabolite 2-methylisoborneol (2-MIB) causes odor and taste issues in drinking water, making it unappealing for human consumption. It has been suggested that 2-MIB biosynthesis consists of two main steps, namely, methylation of geranyl diphosphate into 2-methyl geranyl diphosphate by geranyl diphosphate methyl transferase (GPPMT) and subsequent cyclization into 2-MIB by 2-MIB synthase (MIBS). Pseudanabaena foetida var. intermedia is a 2-MIB-producing cyanobacterium whose GPPMT and MIBS enzymes are encoded by adjacent mtf and mtc genes. The present study identified a 2-MIB-related gene cluster composed of cnbA, mtf, mtc, and cnbB genes in P. foetida var. intermedia. The two homologous cyclic nucleotide-binding protein genes, cnbA and cnbB, were detected adjacent to the mtf and mtc genes, respectively. The nucleotide sequence of the cnbA-mtf-mtc-cnbB gene cluster showed 99.55% identity with 2-MIB synthesis-associated gene cluster of Pseudanabaena sp. dqh15. RT-PCR results revealed that mtf and mtc genes are co-expressed, while cnbA and cnbB genes are expressed independently in P. foetida var. intermedia. To investigate whether only mtf and mtc genes are sufficient for 2-MIB synthesis, the two-gene unit (mtf-mtc) was introduced into Escherichia coli strain JM109 via overexpression vector pYS1C. Gas chromatograph-mass spectrometry results showed that the E. coli strain transformed with mtf-mtc was able to produce 2-MIB. The intracellular 2-MIB level in P. foetida var. intermedia was higher than the extracellular 2-MIB level, while the transformed E. coli strain showed an opposite trend. Growth inhibition was observed in the 2-MIB-producing transformed E. coli strain. IMPORTANCE Contamination of drinking water with odiferous microbial metabolite 2-MIB is a worldwide concern. Removal of 2-MIB from drinking water burdens the water purification process. Therefore, it is important to search for alternative methods, such as suppressing the production of 2-MIB by aquatic microorganisms. For that, it is necessary to expand the current knowledge about the mechanism of 2-MIB synthesis at the genetic level. This study revealed that mtf and mtc genes of the 2-MIB-related gene cluster are transcribed as a single unit in P. foetida var. intermedia, and the expression of both mtf and mtc genes is essential and sufficient for 2-MIB synthesis in E. coli heterologous gene expression system.

Change in expression levels of NAD kinase-encoding genes in Flaveria species.

Nicotinamide adenine dinucleotides (NAD(H)) and NAD phosphates (NADP(H)) are electron carriers involved in redox reactions and metabolic processes in all organisms. NAD kinase (NADK) is the only enzyme that phosphorylates NAD+ into NADP+, using ATP as a phosphate donor. In NADP-dependent malic enzyme (NADP-ME)-type C4 photosynthesis, NADP(H) are required for dehydrogenation by NADP-dependent malate dehydrogenase (NADP-MDH) in mesophyll cells, and decarboxylation by NADP-ME in bundle sheath cells. In this study, we identified five NADK genes (FbNADK1a, 1b, 2a, 2b, and 3) from the C4 model species Flaveria bidentis. RNA-Seq database analysis revealed higher transcript abundance in one of the chloroplast-type NADK2 genes of C4F. bidentis (FbNADK2a). Comparative analysis of NADK activity in leaves of C3, C3-C4, and C4Flaveria showed that C4Flaveria (F. bidentis and F. trinervia) had higher NADK activity than the other photosynthetic-types of Flaveria. Taken together, our results suggest that chloroplastic NAD kinase appeared to increase in importance as C3 plants evolved into C4 plants in the genus Flaveria.

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

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

Physiological Significance of NAD Kinases in Cyanobacteria.

Unicellular cyanobacteria are thought to be the evolutionary ancestors of plant chloroplasts and are widely used both for chemical production and as model organisms in studies of photosynthesis. Although most research focused on increasing reducing power (that is, NADPH) as target of metabolic engineering, the physiological roles of NAD(P)(H) in cyanobacteria poorly understood. In cyanobacteria such as the model species Synechocystis sp. PCC 6803, most metabolic pathways share a single compartment. This complex metabolism raises the question of how cyanobacteria control the amounts of the redox pairs NADH/NAD+ and NADPH/NADP+ in the cyanobacterial metabolic pathways. For example, photosynthetic and respiratory electron transport chains share several redox components in the thylakoid lumen, including plastoquinone, cytochrome b6f (cyt b6f), and the redox carriers plastocyanin and cytochrome c6. In the case of photosynthesis, NADP+ acts as an important electron mediator on the acceptor-side of photosystem I (PSI) in the linear electron chain as well as in the plant chloroplast. Meanwhile, in respiration, most electrons derived from NADPH and NADH are transferred by NAD(P)H dehydrogenases. Therefore, it is expected that Synechocystis employs unique NAD(P)(H) -pool control mechanisms to regulate the mixed metabolic systems involved in photosynthesis and respiration. This review article summarizes the current state of knowledge of NAD(P)(H) metabolism in Synechocystis. In particular, we focus on the physiological function in Synechocystis of NAD kinase, the enzyme that phosphorylates NAD+ to NADP+.

Metabolomic analysis of NAD kinase-deficient mutants of the cyanobacterium Synechocystis sp. PCC 6803.

NAD kinase (NADK) phosphorylates NAD(H) to NADP(H). The enzyme has a crucial role in the regulation of the NADP(H)/NAD(H) ratio in various organisms. The unicellular cyanobacterium Synechocystis sp. PCC 6803 possesses two NADK-encoding genes, sll1415 and slr0400. To elucidate the metabolic change in NADK-deficient mutants growing under photoautotrophic conditions, we conducted metabolomic analysis using capillary electrophoresis mass spectrometry (CE-MS). The growth curves of the wild-type parent (WT) and NADK-deficient mutants (Δ1415 and Δ0400) did not show any differences under photoautotrophic conditions. The NAD(P)(H) balance showed abnormality in both mutants. However, only the metabolite pattern of Δ0400 showed differences compared to WT. These results indicated that the two NADK isoforms have distinct functions in cyanobacterial metabolism.