Elongation Region Research Articles

Genome-Wide Identification and Expression Analysis of the Cys2His2 Zinc Finger Protein Gene Family in Flammulina filiformis.

Zinc finger proteins (ZFPs) are essential transcription factors in eukaryotes, particularly the extensively studied C2H2 family, which is known for its involvement in various biological processes. This research provides a thorough examination and analysis of the C2H2-ZFP gene family in Flammulina filiformis. Using bioinformatics tools, 58 FfC2H2-ZFP genes spread across 11 chromosomes were identified and scrutinized in detail for their gene structures, protein characteristics, and phylogenetic relationships. The study of phylogenetics and synteny sheds light on the evolutionary relationships among C2H2-ZFPs in F. filiformis and other fungi, revealing a complex evolutionary past. The identification of conserved cis-regulatory elements in the gene promoter regions suggests intricate functionalities, particularly in the developmental and stress response pathways. By utilizing RNA-seq and qRT-PCR techniques, the expression patterns of these genes were explored across different developmental stages and tissues of F. filiformis, unveiling distinct expression profiles. Notably, significant expression variations were observed in the stipe elongation region and pilei of various sizes, indicating potential roles in fruiting body morphogenesis. This study enhances our knowledge of the C2H2-ZFP gene family in F. filiformis and lays the groundwork for future investigations into their regulatory mechanisms and applications in fungal biology and biotechnology.

Physiological and transcriptome analysis of changes in endogenous hormone and sugar content during the formation of tender asparagus stems

Asparagus is a nutritionally dense stem vegetable whose growth and development are correlated with its quality and yield. To investigate the dynamic changes and underlying mechanisms during the elongation and growth process of asparagus stems, we documented the growth pattern of asparagus and selected stem segments from four consecutive elongation stages using physiological and transcriptome analyses. Notably, the growth rate of asparagus accelerated at a length of 25 cm. A significant decrease in the concentration of sucrose, fructose, glucose, and additional sugars was observed in the elongation region of tender stems. Conversely, the levels of auxin and gibberellins(GAs) were elevated along with increased activity of enzymes involved in sucrose degradation. A significant positive correlation existed between auxin, GAs, and enzymes involved in sucrose degradation. The ABA content gradually increased with stem elongation. The tissue section showed that cell elongation is an inherent manifestation of stem elongation. The differential genes screened by transcriptome analysis were enriched in pathways such as starch and sucrose metabolism, phytohormone synthesis metabolism, and signal transduction. The expression levels of genes such as ARF, GA20ox, NCED, PIF4, and otherswere upregulated during stem elongation, while DAO, GA2ox, and other genes were downregulated. The gene expression level was consistent with changes in hormone content and influenced the cell length elongation. Additionally, the expression results of RT-qPCR were consistent with RNA-seq. The observed variations in gene expression levels, endogenous hormones and sugar changes during the elongation and growth of asparagus tender stems offer valuable insights for future investigations into the molecular mechanisms of asparagus stem growth and development and provide a theoretical foundation for cultivation and production practices.

Bilirubin Distribution in Plants at the Subcellular and Tissue Levels.

In heterotrophs, heme degradation produces bilirubin, a tetrapyrrole compound that has antioxidant activity. In plants, heme is degraded in plastids and is believed to be converted to phytochromobilin rather than bilirubin. Recently, we used the bilirubin-inducible fluorescent protein UnaG to reveal that plants produce bilirubin via a non-enzymatic reaction with NADPH. In the present study, we used an UnaG-based live imaging system to visualize bilirubin accumulation in Arabidopsis thaliana and Nicotiana benthamiana at the organelle and tissue levels. In chloroplasts, bilirubin preferentially accumulated in the stroma, and the stromal bilirubin level increased upon dark treatment. Investigation of intracellular bilirubin distribution in leaves and roots showed that it accumulated mostly in plastids, with low levels detected in the cytosol and other organelles, such as peroxisomes, mitochondria and the endoplasmic reticulum. A treatment that increased bilirubin production in chloroplasts decreased the bilirubin level in peroxisomes, implying that a bilirubin precursor is transported between the two organelles. At the cell and tissue levels, bilirubin showed substantial accumulation in the root elongation region but little or none in the root cap and guard cells. Intermediate bilirubin accumulation was observed in other shoot and root tissues, with lower levels in shoot tissues. Our data revealed the distribution of bilirubin in plants, which has implications for the transport and physiological function of tetrapyrroles.

Functional divergence of two arginine decarboxylase genes in tropane alkaloid biosynthesis and root growth in Atropa belladonna

Putrescine, produced via the arginine decarboxylase (ADC)/ornithine decarboxylase (ODC)-mediated pathway, is an initial precursor for polyamines metabolism and the root-specific biosynthesis of medicinal tropane alkaloids (TAs). These alkaloids are widely used as muscarinic acetylcholine antagonists in clinics. Although the functions of ODC in biosynthesis of polyamines and TAs have been well investigated, the role of ADC is still poorly understood. In this study, enzyme inhibitor treatment showed that ADC was involved in the biosynthesis of putrescine-derived metabolites and root growth in Atropa belladonna. Further analysis found that there were six ADC unigenes in the A. belladonna transcriptome, with two of them, AbADC1 and AbADC2, exhibiting high expression in the roots. To investigate their roles in TAs/polyamines metabolism and root growth, RNA interference (RNAi) was used to suppress either AbADC1 or AbADC2 expression in A. belladonna hairy roots. Suppression of the AbADC1 expression resulted in a significant reduction in the putrescine content and hairy root biomass. However, it had no noticeable effect on the levels of N-methylputrescine and the TAs hyoscyamine, anisodamine, and scopolamine. On the other hand, suppression of AbADC2 expression markedly reduced the levels of putrescine, N-methylputrescine, and TAs, but had no significant effect on hairy root biomass. According to β-glucuronidase (GUS) staining assays, AbADC1 was mainly expressed in the root elongation and division region while AbADC2 was mainly expressed in the cylinder of the root maturation region. These differences in expression led to functional divergence, with AbADC1 primarily regulating root growth and AbADC2 contributing to TA biosynthesis.

Unusual Stress Upturn in Elastomers Prepared Using Macro Cross-Linkers with Multiple Vinyl Side Groups.

In this study, the unique tensile properties of acrylate elastomers prepared using macro cross-linker polymers with multiple vinyl side groups are analyzed. For the preparation of the macro cross-linker, poly(ethyl acrylate) copolymers bearing hydroxy functional groups are synthesized, followed by the hydroxy-isocyanate reaction with 2-isocyanatoethyl acrylate. Subsequently, the elastomers samples are prepared by UV polymerization of ethyl acrylate in the presence of the macro cross-linkers. The tensile properties of the elastomers in the small elongation region are similar to those of typical elastomers prepared using divinyl cross-linkers, whereas the stress upturn in the large elongation region is considerably different. The stress upturn varies based on the fraction of vinyl side groups in the macro cross-linkers, whereas stress in the small elongation region remains unchanged. These properties are analyzed using various theoretical models. The results reveal that there is artificial inhomogeneity in the cross-link density for samples prepared by the macro cross-linkers, where the short poly(ethyl acrylate) strands inside the macro cross-linker limit the overall chain stretchability. On the whole, this study demonstrates a new method for tuning elastomer properties, especially at large deformation.

Transcriptome Profiling Reveals Candidate Genes Related to Stipe Gradient Elongation of Flammulina filiformis.

Stipe gradient elongation is an important and remarkable feature in the development of most mushroom fruiting bodies. However, its molecular mechanism has rarely been described. Here, the decreasing trend of stipe elongation and increasing trend of cell length in a gradient from the top to the base of the stipe were determined in a model basidiomycete mushroom: Flammulina filiformis. According to RNA-seq results, 1409 differentially expressed genes (DEGs) were identified among elongation region (ER), transition region (TR), and stable region (SR) samples, including 26 transcription factors (TFs). Based on Short Time-series Expression Miner (STEM) clustering of DEGs, clusters 1 and 3, with obvious expression trends that were consistent with or in contrast to the elongation rate, were screened. The cluster 1 DEGs were mainly involved in the GO cellular component category and KEGG genetic information processing class; however, the cluster 3 DEGs were mainly involved in metabolic processes. Furthermore, qRT-PCR confirmed that key genes of the long-chain fatty acid synthesis pathway were involved in stipe gradient elongation and regulated by NADPH oxidase-derived ROS signaling molecules. These findings provide an essential basis for understanding the molecular mechanism of stipe gradient elongation.

Study of the Dynamic Viscoelasticity of Single Poly(N-isopropylacrylamide) Chains Using Atomic Force Microscopy

Thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) is of great interest in the fields of medicine and pharmacology because its lower critical solubility temperature (LCST) is close to the physiological temperature. The understanding of the phase transfer mechanism of PNIPAM near the LCST is of great significance for the design, development, and application of its derivatives. In this study, a dynamic single-molecule force spectroscopy (SMFS) approach was used to quantitatively assess the dynamic viscoelasticity of single PNIPAM chains at different temperatures. We found that the relationship between the viscoelasticity coefficient of a single polymer chain in the low elongation region and the number of chain segments below the LCST was in accordance with the prediction of the Kirkwood model. Above the LCST, the PNIPAM chains exhibited different viscoelastic behaviors that were determined by their conformations. Importantly, the characterization of dynamic viscoelasticity allowed us to observe the phase transition behavior of single polymer chains in the low elongation region where they are similar to random coils, which helped us to understand the microscopic mechanism of their temperature response. In particular, above the LCST, the PNIPAM chains no longer underwent purely entropic elastic behavior but instead displayed enthalpic viscoelastic behavior dominated by intramolecular hydrogen bonds.

Elongation effect on beta-induced Alfvén eigenmode

Beta induced Alfvén eigenmode (BAE) can be an important candidate for ion loss in burning plasmas. Elongation effect on BAE has been investigated by the gyrokinetic eigenvalue code DAEPS in this work. We construct a shaped equilibrium model by modifying local s − α model with which the capability of the DAEPS code has been extended to study the elongation effect. It is discovered that the BAE growth rate first increases with elongation factor κ, reaches a maximum and then decreases. This trend occurs for many different values of η i. We find that, in the weak or moderate elongation region, the BAE instability is reactive type and mainly determined by the fluid/MHD effects, namely the combination of stabilizing field line bending term and destabilizing interchange drive in the vorticity equation. However, in the strong elongation region, the BAE instability becomes dissipative and is mainly driven by the wave–particle resonance effect embedded in δW k since the fluid driving damps away. It is also discovered that the wave–particle resonance decreases with elongation in this region, which is due to the decrease of the geodesic curvature with elongation and leads to the decrease in the growth rate of BAE.

Reactive Oxygen Species Distribution Involved in Stipe Gradient Elongation in the Mushroom Flammulina filiformis.

The mushroom stipe raises the pileus above the substrate into a suitable position for dispersing spores. The stipe elongates at different speeds along its length, with the rate of elongation decreasing in a gradient from the top to the base. However, the molecular mechanisms underlying stipe gradient elongation are largely unknown. Here, we used the model basidiomycete mushroom Flammulina filiformis to investigate the mechanism of mushroom stipe elongation and the role of reactive oxygen species (ROS) signaling in this process. Our results show that O2− and H2O2 exhibit opposite gradient distributions in the stipe, with higher O2− levels in the elongation region (ER), and higher H2O2 levels in the stable region (SR). Moreover, NADPH-oxidase-encoding genes are up-regulated in the ER, have a function in producing O2−, and positively regulate stipe elongation. Genes encoding manganese superoxide dismutase (MnSOD) are up-regulated in the SR, have a function in producing H2O2, and negatively regulate stipe elongation. Altogether, our data demonstrate that ROS (O2−/H2O2) redistribution mediated by NADPH oxidase and MnSODs is linked to the gradient elongation of the F. filiformis stipe.

Ribosome Profiling Reveals Genome-Wide Cellular Translational Regulation in Lacticaseibacillus rhamnosus ATCC 53103 under Acid Stress.

During fermentation and food processing, Lacticaseibacillus rhamnosus ATCC 53103 can encounter many adverse conditions, and acid stress is one of them. The purpose of the present study was to investigate the influence of acid stress on the global translational and transcriptional regulation of Lacticaseibacillus rhamnosus ATCC 53103. Two pH values (pH 6.0 vs. pH 5.0) were applied, the effects of which were studied via ribosome profiling and RNA sequencing assay. Under acid stress, many genes showed differential changes at the translational and transcriptional levels. A total of 10 genes showed different expression trends at the two levels. The expression of 337 genes—which mainly participated in the ABC transporters, amino acid metabolism, and ribosome functional group assembly pathways—was shown to be regulated only at the translational level. The translational efficiency of a few genes participating in the pyrimidine and amino acid metabolism pathways were upregulated. Ribosome occupancy data suggested that ribosomes accumulated remarkably in the elongation region of open reading frame regions under acid stress. This study provides new insights into Lacticaseibacillus rhamnosus ATCC 53103 gene expression under acid stress, and demonstrates that the bacterium can respond to acid stress with synergistic translational and transcriptional regulation mechanisms, improving the vitality of cells.

Analysis and modeling of modified styrene–acrylonitrile/carboxylated acrylonitrile butadiene rubber nanocomposites filled with graphene and graphene oxide: Interfacial interaction and nonlinear elastoplastic behavior

AbstractNonlinear elastoplastic behavior of the nanocomposites based on the styrene–acrylonitrile/carboxylated acrylonitrile butadiene rubber (SAN/XNBR) blend was investigated using experimental and theoretical analysis. Graphene, graphene oxide nanoparticles, and glycidyl methacrylate‐grafted‐XNBR (XNBR‐g‐GMA) as a compatibilizer were incorporated in the SAN/XNBR blends. In this regard, the focus of this study is on modeling of the stress–strain behavior of these nanocomposites, considering the effect of the interfacial interactions made by compatibilizer. For this purpose, field emission scanning electron microscopy (FESEM) and transmission electron microscope (TEM) techniques were used to investigate the relationship between microstructure and mechanical properties of nanocomposites. In addition, FESEM and TEM images showed that the presence of a compatibilizer could influence the dispersion and localization of the nanoparticles. According to the tensile test results, the presence of the compatibilizer increased the mechanical properties of the nanocomposites, specifically elongation at break. Considering the nanocomposite containing compatibilizer and graphene oxide, the elongation at break increased about 570% compared with the nanocomposite without compatibilizer. Better dispersion of graphene oxide and the creation of chemical interaction among components in the presence of the XNBR‐g‐GMA compatibilizer could be the reasons for these improvements, as confirmed by TEM. The usage of the Bergstrom–Boyce model for analyzing the nonlinear elastoplastic behavior of the nanocomposites illustrated proper conformity with the experimental data in the elastic region. However, there are some deviations in the viscoplastic region, particularly close to the breaking elongation region.

A method to increase line-density of grating based on PDMS stretching and PUA replication process

Grating plays an important role in many fields, so it is of great significance to develop a low-cost and rapid grating fabricating technology. In this paper, the stretching deformation process of PDMS is firstly studied by the COMSOL finite element simulation, and the simulation result proves theoretically that the line-density of PDMS grating could be effectively increased by stretching. Then, we propose a novel method to increase the line-density of grating based on PDMS stretching and PUA replication. The relationship between the elongation and uniform region, line-density increase ratio, groove depth reduction is discussed. The experimental results are consistent with the simulation result. The grating diffraction experiment also proves the effectiveness of the method. Because the process can be repeated several times, the line-density of the grating can be further increased. The experimental results show that the grating period decreased from 10 μm to 5 μm after stretching and replication three times. Besides, the process is also applied to the fabrication of high-density grating. Although this process will lead to some loss of grating contour, compared with other grating fabricating technologies, this process has the advantages of low-cost, simplicity, and rapid fabrication. Therefore, it has good application value in the field of grating fabrication.

LYSINE KETOGLUTARATE REDUCTASE TRANS-SPLICING RELATED 1 is involved in temperature-dependent root growth in rice.

Root length is an important root parameter directly related to the uptake of water and nutrients. However, the molecular mechanisms controlling root length are still not fully understood. Here, we isolated a short-root mutant of rice, dice2 (defective in cell elongation 2). The cell length and meristem size of the roots were decreased in dice2, but the root function in terms of mineral element uptake, root cell width, and root anatomy were hardly altered compared with wild-type (WT) rice. The root growth defect in dice2 could be partially rescued by high temperature. Map-based cloning combined with a complementation test revealed that the short-root phenotype was caused by a nonsense mutation in a gene which was annotated to encode Lysine Ketoglutarate Reductase Trans-Splicing related 1 (OsLKRT1). OsLKRT1, encoding a cytosol-localized protein, was expressed in all cells of the root tip and elongation region as well as the shoot. RNA-seq analysis showed that there was no difference between dice2 and the WT in the expression level of genes involved in root development identified so far. These results indicate that OsLKRT1 is involved in a novel pathway required for root cell elongation in rice, although its exact role remains to be further investigated.

Effects of drought stress on shoot development of tomato (Solanum lycopersicum L.)

In recent years, drought stress was strongly affected on the development and yield of tomatoes. There are increasing interests in the study of physiological transformations in adaption to stress in plants In this study, effects of drought stress (mannitol at different concentration) on the development of tomato shoot were studied. Morphological and physiological changes during the development of shoot under drought stress conditions were analyzed. Based on the analysis results, the combination of cytokinin and gibberellin was treated to increase the drought stress tolerance of plants. Results showed that mannitol at 20 g/L induced tomato drought stress. Shoot height, number of leaves, leaf area, and the number of roots significantly decreased in the drought stress condition compared to the control. The formation superoxide (O2-) and hydrogen peroxide (H2O2) occurred in the meristem, elongation region and cap of the roots in the drought stress condition instead of only cap root in the control. In the drought stress condition, there was an increase in respiration intensity, proline and carotenoid content, and abscisic acid activity. In contrast, the content of chlorophyll, photosynthesis intensity, cytokinin and gibberellin activity decreased in comparison with the control. The combination treatment of zeatin 0.5 mg/L and GA3 0.5 mg/L improved the drought stress tolerance of plants. The shoot height, number of leaves, leaf area and number of roots of the treated plants were higher than those of the control plants.

Transcriptome dynamic landscape underlying the improvement of maize lodging resistance under coronatine treatment

BackgroundLodging is one of the important factors causing maize yield. Plant height is an important factor in determining plant architecture in maize (Zea mays L.), which is closely related to lodging resistance under high planting density. Coronatine (COR), which is a phytotoxin and produced by the pathogen Pseudomonas syringae, is a functional and structural analogue of jasmonic acid (JA).ResultsIn this study, we found COR, as a new plant growth regulator, could effectively reduce plant height and ear height of both hybrids (ZD958 and XY335) and inbred (B73) maize by inhibiting internode growth during elongation, thus improve maize lodging resistance. To study gene expression changes in internode after COR treatment, we collected spatio-temporal transcriptome of inbred B73 internode under normal condition and COR treatment, including the three different regions of internode (fixed, meristem and elongation regions) at three different developmental stages. The gene expression levels of the three regions at normal condition were described and then compared with that upon COR treatment. In total, 8605 COR-responsive genes (COR-RGs) were found, consist of 802 genes specifically expressed in internode. For these COR-RGs, 614, 870, 2123 of which showed expression changes in only fixed, meristem and elongation region, respectively. Both the number and function were significantly changed for COR-RGs identified in different regions, indicating genes with different functions were regulated at the three regions. Besides, we found more than 80% genes of gibberellin and jasmonic acid were changed under COR treatment.ConclusionsThese data provide a gene expression profiling in different regions of internode development and molecular mechanism of COR affecting internode elongation. A putative schematic of the internode response to COR treatment is proposed which shows the basic process of COR affecting internode elongation. This research provides a useful resource for studying maize internode development and improves our understanding of the COR regulation mechanism based on plant height.

Differential responses of roots for varying tolerance to salinity stress in wheat with special reference to elasticity

Two salt-sensitive (Yongliang-15, GS-6058) and two salt-tolerant (JS-7, Xinchun-31) wheat cultivars were used to investigate the extension, extensibility (viscoelastic parameters), and chemical composition of the cell walls in their root elongation regions (apical 10 mm-long root segments), under salinity stress. The elasticity of the root cell wall, indicated by E0, significantly decreased in the salt-sensitive cultivars, whereas the E0 in the salt-tolerant cultivars was maintained at the same level as that in the non-saline condition. Root extension and the differences among cultivars were largely dependent on elastic extension, which accounts for one-half to two-thirds of the total extension. Viscosity, indicated by η0, and the plastic extension of the root cell walls did not change across the treatments and cultivars. The significant decrease in cell wall elasticity in the root elongation region was one of the factors that depressed root growth in salt-sensitive cultivars under the saline condition. The well-maintained elasticity of salt-tolerant cultivars alleviated the depression of root growth by NaCl. Cell wall elasticity was positively correlated with the relative pectin and hemicellulose I contents and negatively correlated with the relative cellulose content. Under saline conditions, the relative hemicellulose II content did not change in the salt-sensitive cultivars; however, it decreased in the salt-tolerant ones. Thus, changes in chemical composition of the cell wall were correspond with the cell wall extensibility and root growth in wheat cultivars with different degrees of salt tolerance.

NaCl-altered oxygen flux profiles and H+-ATPase activity in roots of two contrasting poplar species.

Maintaining mitochondrial respiration is crucial for proving ATP for H+ pumps to continuously exclude Na+ under salt stress. NaCl-altered O2 uptake, mitochondrial respiration and the relevance to H+-ATPase activity were investigated in two contrasting poplar species, Populus euphratica (salt-tolerant) and Populus popularis 35-44 (salt-sensitive). Compared with P. popularis, P. euphratica roots exhibited a greater capacity to extrude Na+ under NaCl stress (150mM). The cytochemical analysis with Pb(NO3)2 staining revealed that P. euphratica root cells retained higher H+ hydrolysis activity than the salt-sensitive poplar during a long term (LT) of increasing salt stress (50-200mM NaCl, 4weeks). Long-sustained activation of proton pumps requires long-lasting supply of energy (adenosine triphosphate, ATP), which is delivered by aerobic respiration. Taking advantage of the vibrating-electrodes technology combined with the use of membrane-tipped, polarographic oxygen microelectrodes, the species, spatial and temporal differences in root O2 uptake were characterized under conditions of salt stress. Oxygen uptake upon NaCl shock (150mM) was less declined in P. euphratica than in P. popularis, although the salt-induced transient kinetics were distinct from the drastic drop of O2 caused by hyperosmotic shock (255mM mannitol). Short-term (ST) treatment (150mM NaCl, 24h) stimulated O2 influx in P. euphratica roots, and LT-treated P. euphratica displayed an increased O2 influx along the root axis, whereas O2 influx declined with increasing salinity in P. popularis roots. The spatial localization of O2 influxes revealed that the apical zone was more susceptible than the elongation region upon high NaCl (150, 200mM) during ST and LT stress. Pharmacological experiments showed that the Na+ extrusion and H+-ATPase activity in salinized roots were correspondingly suppressed when O2 uptake was inhibited by a mitochondrial respiration inhibitor, NaN3. Therefore, we conclude that the stable mitochondrial respiration energized H+-ATPase of P. euphratica root cells for maintaining Na+ homeostasis under salt environments.

Detection of Filamentous Microorganisms in Fluorescence Microscopy Images.

We present a robust, precise image binarization technique for automatically detecting filamentous microorganisms from digital fluorescence microscopy scans, with application to finding the pseudohyphae that are fungal pathogens responsible for Candida vaginitis. This method employs a hybrid constant false positive rate processor that integrates cell average and order statistic detectors, with linear windows at multiple orientation angles. The hypothesis test rule incorporates elongation enhancement and region of interest masking. Our approach achieves the adaptivity to local noise and all possible object orientations. The designed processor is evaluated theoretically and experimentally using clinical images. Successful detection results are demonstrated.

Cross-Shore Deformation of a Surfzone-Released Dye Plume by an Internal Tide on the Inner Shelf

AbstractAn inner-shelf (IS) dye plume that formed following a 3.84-h early morning surfzone (SZ) dye release off of Imperial Beach, California, is analyzed with in situ and aerial remotely sensed observations. Midmorning, 5 h after release start, the IS plume extended 800 m offshore (or ≈8Lsz, where Lsz is the surfzone width) and was surface intensified. Over the next ≈2 h, the IS plume deformed (narrowed) cross-shore with the offshore front progressing onshore at ≈5 cm s−1, deepened by up to 3 m, and elongated alongshore at ≈4.5 cm s−1 km−1 (at ≈2.5Lsz). Coincident with IS plume deformation and deepening, IS isotherms also deepened, with relatively stable IS plume joint dye and temperature statistics. Offshore tracer transport and subsequent IS plume deformation and deepening likely resulted from two phases of the diurnal internal tide (DIT). During and after deformation, the IS plume did not reenter the warm surfzone, which potentially acted as a thermal barrier. High-frequency internal waves (HF IWs) propagated through the IS plume at ≈9 cm s−1 and dissipated onshore of 4Lsz. Surface HF IW signal was elevated in the plume elongation region, suggesting a linkage between plume elongation and either the DIT or HF IW. This IS plume evolution differs from previous SZ tracer releases, highlighting the effects of release timing relative to the solar cycle or the internal tide.

An in Vivo Binding Assay for RNA-Binding Proteins Based on Repression of a Reporter Gene

We study translation repression in bacteria by engineering a regulatory circuit that functions as a binding assay for RNA binding proteins (RBP) in vivo. We do so by inducing expression of a fluorescent protein-RBP chimera, together with encoding its binding site at various positions within the ribosomal initiation region (+11-13 nt from the AUG) of a reporter module. We show that when bound by their cognate RBPs, the phage coat proteins for PP7 (PCP) and Qβ (QCP), strong repression is observed for all hairpin positions within the initiation region. Yet, a sharp transition to no-effect is observed when positioned in the elongation region, at a single-nucleotide resolution. Employing in vivo Selective 2'-hydroxyl acylation analyzed by primer extension followed by sequencing (SHAPE-seq) for a representative construct, established that in the translationally active state the mRNA molecule is nonstructured, while in the repressed state a structured signature was detected. We then utilize this regulatory phenomena to quantify the binding affinity of the coat proteins of phages MS2, PP7, GA, and Qβ to 14 cognate and noncognate binding sites in vivo. Using our circuit, we demonstrate qualitative differences between in vitro to in vivo binding characteristics for various variants when comparing to past studies. Furthermore, by introducing a simple mutation to the loop region for the Qβ-wt site, MCP binding is abolished, creating the first high-affinity QCP site that is completely orthogonal to MCP. Consequently, we demonstrate that our hybrid transcriptional-post-transcriptional circuit can be utilized as a binding assay to quantify RNA-RBP interactions in vivo.