scholarly journals A Tobacco Syringe Agroinfiltration-Based Method for a Phytohormone Transporter Activity Assay Using Endogenous Substrates

2021 ◽  
Vol 12 ◽  
Author(s):  
Jiangzhe Zhao ◽  
Min Ju ◽  
Jiayun Qian ◽  
Mengyuan Zhang ◽  
Ting Liu ◽  
...  
Keyword(s):  
Stress Responses ◽  
Large Scale ◽  
Functional Characterization ◽  
Endogenous Hormones ◽  
Activity Assay ◽  
Long Distance ◽  
Multiple Substrates ◽  
Long Distance Transport ◽  
Endogenous Substrates

Phytohormones are a group of small chemical molecules that play vital roles in plant development, metabolism, and stress responses. Phytohormones often have distinct biosynthesis and signaling perception sites, requiring long- or short-distance transportation. Unlike biosynthesis and signal transduction, phytohormone transport across cells and organs is poorly understood. The transporter activity assay is a bottleneck for the functional characterization of novel phytohormone transporters. In the present study, we report a tobacco syringe agroinfiltration and liquid chromatography tandem mass spectrometry (TSAL)-based method for performing a phytohormone transporter activity assay using endogenous hormones present in tobacco (Nicotiana benthamiana) leaves. A transporter activity assay using this method does not require isotope-labeled substrates and can be conveniently performed for screening multiple substrates by using endogenous hormones in tobacco leaves. The transporter activities of three known hormone transporters, namely AtABCG25 for abscisic acid, AtABCG16 for jasmonic acid, and AtPUP14 for cytokinin, were all successfully validated using this method. Using this method, cytokinins were found to be the preferred substrates of an unknown maize (Zea mays) transporter ZmABCG43. ZmABCG43 transporter activities toward cytokinins were confirmed in a cytokinin long-distance transport mutant atabcg14 through gene complementation. Thus, the TSAL method has the potential to be used for basic substrate characterization of novel phytohormone transporters or for the screening of novel transporters for a specific phytohormone on a large scale.

scholarly journals Genome-wide identification and functional characterization of Camelina sativa WRKY gene family in response to abiotic stress

2020 ◽  
Author(s):  
Yanan Song ◽  
Hongli Cui ◽  
Ying Shi ◽  
Jinai Xue ◽  
Chunli Ji ◽  
...  
Keyword(s):  
Gene Family ◽  
Stress Responses ◽  
Stress Resistance ◽  
Segmental Duplication ◽  
Functional Characterization ◽  
High Stress ◽  
Camelina Sativa ◽  
Genome Wide ◽  
Large Expansion

Abstract Background: WRKY transcription factors are a superfamily of regulators involved in diverse biological processes and stress responses in plants. However, knowledge is limited for WRKY family in camelina (Camelina sativa), an important Brassicaceae oil crop with strong tolerance against various stresses. Here, genome-wide characterization of WRKY proteins is performed to examine their gene-structures, phylogenetics, expressions, conserved motif organizations, and functional annotation to identify candidate WRKYs mediating regulation of stress resistance in camelina.Results: Total of 242 CsWRKY proteins encoded by 224 gene loci distributed uneven on chromosomes were identified, and classified into three groups via phylogenetic analysis according to their WRKY domains and zinc finger motifs. 15 CsWRKY gene loci generated 33 spliced variants. Orthologous WRKY gene pairs were identified, with 173 pairs in C. sativa and Arabidopsis genomes as well as 282 pairs for C. sativa and B. napus, respectively. 137 segmental duplication events were observed but no tandem duplication in camelina genome. Ten major conserved motifs were examined, with WRKYGQK as the most conserved and several variants existed in many CsWRKYs. Expression analysis revealed that half more CsWRKY genes were expressed constitutively, and a set of them had a tissue-specific expression. Notably, 11 CsWRKY genes exhibited significantly expression changes in plant seedlings under cold, salt, and drought stress, respectively, having preferentially inducible expression pattern in response to the stress.Conclusions: The present described a detail analysis of CsWRKY gen family and their expression profiled in twelve tissues and under several stress conditions. Segmental duplication is the major force for large expansion of this gene family, and a strong purifying pressure happened for CsWRKY proteins evolutionally. CsWRKY proteins play important roles for plant development, with differential functions in different tissues. Exceptionally, eleven CsWRKYs, particularly five alternative spliced isoforms were found to be the key players possibly in mediating plant response to various stresses. Overall, our results provide a foundation for understanding roles of CsWRKYs and the precise mechanism through which CsWRKYs regulate high stress resistance to stress as well as development of stress tolerance cultivars for Cruciferae crops.

scholarly journals sRNA Target Prediction Organizing Tool (SPOT) Integrates Computational and Experimental Data To Facilitate Functional Characterization of Bacterial Small RNAs

mSphere ◽  
2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Alisa M. King ◽  
Carin K. Vanderpool ◽  
Patrick H. Degnan
Keyword(s):  
Experimental Data ◽  
Stress Responses ◽  
Small Rnas ◽  
Metabolic Regulation ◽  
Functional Characterization ◽  
Target Prediction ◽  
Structure Stability ◽  
Accurate Identification ◽  
Mrna Targets

Small RNAs (sRNAs) regulate gene expression in diverse bacteria by interacting with mRNAs to change their structure, stability, or translation. Hundreds of sRNAs have been identified in bacteria, but characterization of their regulatory functions is limited by difficulty with sensitive and accurate identification of mRNA targets. Thus, new robust methods of bacterial sRNA target identification are in demand. Here, we describe our small RNA target prediction organizing tool (SPOT), which streamlines the process of sRNA target prediction by providing a single pipeline that combines available computational prediction tools with customizable results filtering based on experimental data. SPOT allows the user to rapidly produce a prioritized list of predicted sRNA-target mRNA interactions that serves as a basis for further experimental characterization. This tool will facilitate elucidation of sRNA regulons in bacteria, allowing new discoveries regarding the roles of sRNAs in bacterial stress responses and metabolic regulation.

scholarly journals Functional characterization of 3D protein structures informed by human genetic diversity

2019 ◽  
Vol 116 (18) ◽  
pp. 8960-8965 ◽  
Author(s):  
Michael Hicks ◽  
Istvan Bartha ◽  
Julia di Iulio ◽  
J. Craig Venter ◽  
Amalio Telenti
Keyword(s):  
Large Scale ◽  
Functional Characterization ◽  
Protein Structures ◽  
Large Scale Data ◽  
Allosteric Sites ◽  
Missense Variation ◽  
Binding Pockets ◽  
Definition Of ◽  
Scale Data

Sequence variation data of the human proteome can be used to analyze 3D protein structures to derive functional insights. We used genetic variant data from nearly 140,000 individuals to analyze 3D positional conservation in 4,715 proteins and 3,951 homology models using 860,292 missense and 465,886 synonymous variants. Sixty percent of protein structures harbor at least one intolerant 3D site as defined by significant depletion of observed over expected missense variation. Structural intolerance data correlated with deep mutational scanning functional readouts for PPARG, MAPK1/ERK2, UBE2I, SUMO1, PTEN, CALM1, CALM2, and TPK1 and with shallow mutagenesis data for 1,026 proteins. The 3D structural intolerance analysis revealed different features for ligand binding pockets and orthosteric and allosteric sites. Large-scale data on human genetic variation support a definition of functional 3D sites proteome-wide.

Functional characterization of a Glycine soja Ca2+ATPase in salt–alkaline stress responses

2016 ◽  
Vol 90 (4-5) ◽  
pp. 419-434 ◽  
Author(s):  
Mingzhe Sun ◽  
Bowei Jia ◽  
Na Cui ◽  
Yidong Wen ◽  
Huizi Duanmu ◽  
...  
Keyword(s):  
Stress Responses ◽  
Glycine Soja ◽  
Functional Characterization ◽  
Alkaline Stress

scholarly journals Crowdsourced RNA design discovers diverse, reversible, efficient, self-contained molecular sensors

2019 ◽  
Author(s):  
Johan O. L. Andreasson ◽  
Michael R. Gotrik ◽  
Michelle J. Wu ◽  
Hannah K. Wayment-Steele ◽  
Wipapat Kladwang ◽  
...  
Keyword(s):  
High Throughput ◽  
Online Community ◽  
Large Scale ◽  
Functional Characterization ◽  
New Paradigm ◽  
Molecular Sensors ◽  
Rna Switches ◽  
Rna Sensors ◽  
Rna Design

AbstractInternet-based scientific communities promise a means to apply distributed, diverse human intelligence towards previously intractable scientific problems. However, current implementations have not allowed communities to propose experiments to test all emerging hypotheses at scale or to modify hypotheses in response to experiments. We report high-throughput methods for molecular characterization of nucleic acids that enable the large-scale videogame-based crowdsourcing of functional RNA sensor design, followed by high-throughput functional characterization. Iterative design testing of thousands of crowdsourced RNA sensor designs produced near-thermodynamically optimal and reversible RNA switches that act as self-contained molecular sensors and couple five distinct small molecule inputs to three distinct protein binding and fluorogenic outputs—results that surpass computational and expert-based design. This work represents a new paradigm for widely distributed experimental bioscience.One Sentence SummaryOnline community discovers standalone RNA sensors.

scholarly journals A robust metaproteomics pipeline for a holistic taxonomic and functional characterization of microbial communities from marine particles

2019 ◽  
Author(s):  
Doreen Schultz ◽  
Daniela Zühlke ◽  
Jörg Bernhardt ◽  
Thomas Ben Francis ◽  
Dirk Albrecht ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Bacterial Communities ◽  
Large Scale ◽  
Functional Characterization ◽  
Phosphorus Uptake ◽  
Free Living ◽  
Technical Problems ◽  
Eukaryotic Microorganisms ◽  
Marine Particles

SummaryThis study aimed to establish a robust, reproducible and reliable metaproteomic pipeline for an in-depth characterization of marine particle-associated (PA) bacteria. To this end, we compared six well-established protein extraction protocols together with different MS-sample preparation techniques using particles sampled during a North Sea spring algae bloom in 2009. In this optimized workflow, proteins are extracted using a combination of SDS-containing lysis buffer and cell disruption by bead-beating, separated by SDS-PAGE, in-gel digested and analysed by LC-MS/MS, before MASCOT search against a metagenome-based database and data processing/visualization with the in-house-developed bioinformatics tools Prophane and Paver.As proof of principle, free-living (FL) and particulate communities sampled in April 2009 were analysed, resulting in an as yet unprecedented number of 9,354 and 5,034 identified protein groups for FL and PA bacteria, respectively. Our data revealed that FL and PA communities appeared similar in their taxonomic distribution, with notable exceptions: eukaryotic proteins and proteins assigned to Flavobacteriia, Cyanobacteria, and some proteobacterial genera were found more abundant on particles, whilst overall proteins belonging to Proteobacteria were more dominant in the FL fraction. In contrast, significant functional differences including proteins involved in polysaccharide degradation, sugar- and phosphorus uptake, adhesion, motility, and stress response were detected.Originality-Significance StatementMarine particles consist of organic particulate matter (e.g. phyto- or zooplankton) and particle-associated (PA) microbial communities, which are often embedded in a sugary matrix. A significant fraction of the decaying algal biomass in marine ecosystems is expected to be mineralized by PA heterotrophic communities, which are thus greatly contributing to large-scale carbon fluxes. Whilst numerous studies have investigated the succession of planktonic marine bacteria along phytoplankton blooms, the community structure and functionality of PA bacterial communities remained largely unexplored and knowledge on specific contributions of these microorganisms to carbon cycling is still surprisingly limited. This has been mostly been due to technical problems, i.e. to the difficulty to retrieve genomic DNA and proteins from these polysaccharide-rich entities, their enormous complexity and the high abundance of eukaryotic microorganisms.Our study presents an innovative, robust, reproducible, and reliable metaproteomics pipeline for marine particles, which will help to address and fill the above-described knowledge gap. Employing the here established workflow enabled us to identify more than 5,000 PA proteins, which is, at least to our knowledge, the largest number of protein groups ever assigned to marine particles. Notably, the novel pipeline has been validated by a first, comparative metaproteome analysis of free-living and PA bacterial communities indicating a significant functional shift enabling surface-associated bacteria to adapt to particle-specific living conditions. In conclusion, our novel metaproteomics pipeline presents a solid and promising methodological groundwork for future culture-independent analyses of seasonal taxonomic and functional successions of PA microbial communities in aquatic habitats.

scholarly journals Molecular Characterization of Auxin Efflux Carrier- ABCB1 in hexaploid wheat

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Amita Mohan ◽  
Amandeep K. Dhaliwal ◽  
Ragupathi Nagarajan ◽  
Kulvinder S. Gill
Keyword(s):  
Promoter Region ◽  
Sequence Similarity ◽  
Regulatory Element ◽  
Long Distance ◽  
Long Distance Transport ◽  
Response Differentiation ◽  
Cell Tissue ◽  
Seed Width ◽  
Auxin Efflux Carrier

AbstractAuxin is an important phytohormone that regulates response, differentiation, and development of plant cell, tissue, and organs. Along with its local production, long-distance transport coordinated by the efflux/influx membrane transporters is instrumental in plant development and architecture. In the present study, we cloned and characterized a wheat (Triticum aestivum) auxin efflux carrier ABCB1. The TaABCB1 was physically localized to the proximal 15% of the short arm of wheat homoeologous group 7 chromosomes. Size of the Chinese spring (CS) homoeologs genomic copies ranged from 5.3–6.2 kb with the 7A copy being the largest due to novel insertions in its third intron. The three homoeologous copies share 95–97% sequence similarity at the nucleotide, 98–99% amino acid, and overall Q-score of 0.98 at 3-D structure level. Though detected in all analyzed tissues, TaABCB1 predominantly expressed in the meristematic tissues likely due to the presence of meristem-specific activation regulatory element identified in the promoter region. RNAi plants of TaABCB1 gene resulted in reduced plant height and increased seed width. Promoter analysis revealed several responsive elements detected in the promoter region including that for different hormones as auxin, gibberellic acid, jasmonic acid and abscisic acid, light, and circadian regulated elements.

scholarly journals Genetic variants of human organic anion transporter 4 demonstrate altered transport of endogenous substrates

2010 ◽  
Vol 299 (4) ◽  
pp. F767-F775 ◽  
Author(s):  
James E. Shima ◽  
Takafumi Komori ◽  
Travis R. Taylor ◽  
Doug Stryke ◽  
Michiko Kawamoto ◽  
...  
Keyword(s):  
Genetic Variants ◽  
Drug Disposition ◽  
Organic Anion ◽  
Functional Characterization ◽  
Organic Anion Transporter ◽  
Loss Of Function ◽  
Anion Transporter ◽  
Endogenous Substrates ◽  
Slc Transporter

Apical reabsorption from the urine has been shown to be important for such processes as the maintenance of critical metabolites in the blood and the excretion of nephrotoxic compounds. The solute carrier (SLC) transporter OAT4 ( SLC22A11) is expressed on the apical membrane of renal proximal tubule cells and is known to mediate the transport of a variety of xenobiotic and endogenous organic anions. Functional characterization of genetic variants of apical transporters thought to mediate reabsorption, such as OAT4, may provide insight into the genetic factors influencing the complex pathways involved in drug elimination and metabolite reclamation occurring in the kidney. Naturally occurring genetic variants of OAT4 were identified in public databases and by resequencing DNA samples from 272 individuals comprising 4 distinct ethnic groups. Nine total nonsynonymous variants were identified and functionally assessed using uptake of three radiolabeled substrates. A nonsense variant, R48Stop, and three other variants (R121C, V155G, and V155M) were found at frequencies of at least 2% in an ethnic group specific fashion. The L29P, R48Stop, and H469R variants displayed a complete loss of function, and kinetic analysis identified a reduced Vmax in the common nonsynonymous variants. Plasma membrane levels of OAT4 protein were absent or reduced in the nonfunctional variants, providing a mechanistic reason for the observed loss of function. Characterization of the genetic variants of reabsorptive transporters such as OAT4 is an important step in understanding variability in tubular reabsorption with important implications in innate homeostatic processes and drug disposition.

Sign in / Sign up

Export Citation Format

Share Document