Tandem Fluorescent Protein Timers for Noninvasive Relative Protein Lifetime Measurement in Plants

Tandem fluorescent protein timers enable non-invasive analysis of protein turnover in intact plant cells, allowing tests of how different genetic backgrounds and treatments affect protein stability. Targeted protein degradation is an important and pervasive regulatory mechanism in plants, required for perception and response to the environment as well as developmental signaling. Despite the significance of this process, relatively few studies have assessed plant protein turnover in a quantitative fashion. Tandem fluorescent protein timers (tFTs) offer a powerful approach for the assessment of in vivo protein turnover in distinct subcellular compartments of single or multiple cells. A tFT is a fusion of two different fluorescent proteins with distinct fluorophore maturation kinetics, which enable protein age to be estimated from the ratio of fluorescence intensities of the two fluorescent proteins. Here, we used short-lived auxin signaling proteins and model N-end rule (N-recognin) pathway reporters to demonstrate the utility of tFTs for studying protein turnover in living plant cells of Arabidopsis (Arabidopsis thaliana) and Nicotiana benthamiana. We present transient expression of tFTs as an efficient screen for relative protein lifetime, useful for testing the effects of mutations and different genetic backgrounds on protein stability. This work demonstrates the potential for using stably expressed tFTs to study native protein dynamics with high temporal resolution in response to exogenous or endogenous stimuli.

• Publication year

  2019

• Venue

  Plant Physiology

• Publication date

  2019-03-14

• Fields of study

  Biology, Medicine, Chemistry, Environmental Science

• Identifiers
  DOI 10.1104/pp.19.00051PMID 30872425PMCID 6548237
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• Mapping Degradation Signals and Pathways in a Eukaryotic N-terminome.

  2018cited by this paper
• Yeast cells contain a heterogeneous population of peroxisomes that segregate asymmetrically during cell division

  2018cited by this paper
• Photoreceptor-mediated regulation of the COP1/SPA E3 ubiquitin ligase.

  2018cited by this paper
• Multiplexed Proteome Dynamics Profiling Reveals Mechanisms Controlling Protein Homeostasis

  2018cited by this paper
• Bicoid gradient formation mechanism and dynamics revealed by protein lifetime analysis

  2018cited by this paper
• Single Live Cell Monitoring of Protein Turnover Reveals Intercellular Variability and Cell-Cycle Dependence of Degradation Rates.

  2018cited by this paper
• Ubiquitylation in plants: signaling hub for the integration of environmental signals

  2018cited by this paper
• N-terminal acetylation: an essential protein modification emerges as an important regulator of stress responses.

  2018cited by this paper
• Real‐time detection of N‐end rule‐mediated ubiquitination via fluorescently labeled substrate probes

  2017cited by this paper
• Protein Degradation Rate in Arabidopsis thaliana Leaf Growth and Development[OPEN]

  2017cited by this paper
• N‐terminomics reveals control of Arabidopsis seed storage proteins and proteases by the Arg/N‐end rule pathway

  2017cited by this paper
• TimerQuant: a modelling approach to tandem fluorescent timer design and data interpretation for measuring protein turnover in embryos

  2016cited by this paper
• Time-resolved Analysis of Proteome Dynamics by Tandem Mass Tags and Stable Isotope Labeling in Cell Culture (TMT-SILAC) Hyperplexing*

  2016cited by this paper
• From start to finish: amino-terminal protein modifications as degradation signals in plants.

  2016cited by this paper
• Protein turnover measurement using selected reaction monitoring-mass spectrometry (SRM-MS)

  2016cited by this paper
• Rate Motifs Tune Auxin/Indole-3-Acetic Acid Degradation Dynamics1[OPEN]

  2015cited by this paper
• The logic of communication: roles for mobile transcription factors in plants.

  2015cited by this paper
• Two N-Terminal Acetyltransferases Antagonistically Regulate the Stability of a Nod-Like Receptor in Arabidopsis

  2015cited by this paper
• Downregulation of N-terminal acetylation triggers ABA-mediated drought responses in Arabidopsis

  2015cited by this paper
• New insights into root gravitropic signalling.

  2015cited by this paper
• Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers

  2015influential reference
• Protein turnover in plant biology

  2015influential reference
• Agrobacterium tumefaciens-mediated transient transformation of Arabidopsis thaliana leaves.

  2014cited by this paper
• The eukaryotic N-end rule pathway: conserved mechanisms and diverse functions.

  2014cited by this paper
• Intercellular protein movement: deciphering the language of development.

  2014cited by this paper
• Analysis of protein dynamics with tandem fluorescent protein timers.

  2014influential reference
• Tracking protein turnover and degradation by microscopy: photo-switchable versus time-encoded fluorescent proteins

  2014cited by this paper
• Quantitative analysis of protein turnover in plants

  2014influential reference
• Time-profiling fluorescent reporters in the Arabidopsis root.

  2014cited by this paper
• PIN-driven polar auxin transport in plant developmental plasticity: a key target for environmental and endogenous signals.

  2014cited by this paper
• Degradation rate of mitochondrial proteins in Arabidopsis thaliana cells.

  2013cited by this paper
• Intracellular protein degradation: from a vague idea through the lysosome and the ubiquitin-proteasome system and onto human diseases and drug targeting.

  2013cited by this paper
• Lateral root morphogenesis is dependent on the mechanical properties of the overlaying tissues

  2013cited by this paper
• Directional tissue migration through a self-generated chemokine gradient

  2013influential reference
• Tandem fluorescent protein timers for in vivo analysis of protein dynamics

  2012influential reference
• Sequence- and species-dependence of proteasomal processivity.

  2012cited by this paper
• A monomeric red fluorescent protein with low cytotoxicity

  2012cited by this paper
• A combinatorial TIR1/AFB-Aux/IAA co-receptor system for differential sensing of auxin

  2012cited by this paper
• A novel sensor to map auxin response and distribution at high spatio-temporal resolution

  2012cited by this paper
• IAA8 Involved in Lateral Root Formation Interacts with the TIR1 Auxin Receptor and ARF Transcription Factors in Arabidopsis

  2012cited by this paper
• Proteome Dynamics: Revisiting Turnover with a Global Perspective*

  2012cited by this paper
• Mass spectrometry-based proteomics and network biology.

  2012cited by this paper
• The N‐end rule pathway and regulation by proteolysis

  2011influential reference
• A Quantitative Spatial Proteomics Analysis of Proteome Turnover in Human Cells*

  2011cited by this paper
• Homeostatic response to hypoxia is regulated by the N-end rule pathway in plants

  2011cited by this paper
• Oxygen sensing in plants is mediated by an N-end rule pathway for protein destabilization

  2011cited by this paper
• Infiltration of Nicotiana benthamiana Protocol for Transient Expression via Agrobacterium

  2011cited by this paper
• Proteome Half-Life Dynamics in Living Human Cells

  2011cited by this paper
• Global quantification of mammalian gene expression control

  2011cited by this paper
• The cullin-RING ubiquitin-protein ligases.

  2011cited by this paper
• A novel aux/IAA28 signaling cascade activates GATA23-dependent specification of lateral root founder cell identity.

  2010cited by this paper
• Structure and Function of the Hetero-oligomeric Cysteine Synthase Complex in Plants*

  2010cited by this paper
• N-Terminal Acetylation of Cellular Proteins Creates Specific Degradation Signals

  2010cited by this paper
• Subcellular trafficking of PIN auxin efflux carriers in auxin transport.

  2010cited by this paper
• The ubiquitin–26S proteasome system at the nexus of plant biology

  2009influential reference
• Turnover of the human proteome: determination of protein intracellular stability by dynamic SILAC.

  2009cited by this paper
• The N-end rule pathway controls multiple functions during Arabidopsis shoot and leaf development

  2009influential reference
• Root System Architecture from Coupling Cell Shape to Auxin Transport

  2008cited by this paper
• Corresponding authors.

  2008cited by this paper
• Overexpression of the non-canonical Aux/IAA genes causes auxin-related aberrant phenotypes in Arabidopsis.

  2008cited by this paper
• PRT6/At5g02310 encodes an Arabidopsis ubiquitin ligase of the N‐end rule pathway with arginine specificity and is not the CER3 locus

  2007influential reference
• Building Blocks for Plant Gene Assembly1[W][OA]

  2007cited by this paper
• Nuclear localization of enhanced green fluorescent protein homomultimers.

  2007cited by this paper
• Transcriptional and posttranscriptional regulation of transcription factor expression in Arabidopsis roots.

  2006cited by this paper
• Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants

  2006cited by this paper
• The Arabidopsis Aux/IAA Protein Family Has Diversified in Degradation and Auxin Responsiveness[W]

  2006influential reference
• Plant development is regulated by a family of auxin receptor F box proteins.

  2005cited by this paper
• Immunopurification of Polyribosomal Complexes of Arabidopsis for Global Analysis of Gene Expression1[w]

  2005cited by this paper
• Root gravitropism requires lateral root cap and epidermal cells for transport and response to a mobile auxin signal

  2005cited by this paper
• Plant Development Is Regulated by a Family of Auxin Receptor F Box Proteins

  2005cited by this paper
• Modular cloning in plant cells.

  2005cited by this paper
• RAC GTPases in Tobacco and Arabidopsis Mediate Auxin-Induced Formation of Proteolytically Active Nuclear Protein Bodies That Contain AUX/IAA Proteinsw⃞

  2005cited by this paper
• The F-box protein TIR1 is an auxin receptor

  2005cited by this paper
• The Arabidopsis F-box protein TIR1 is an auxin receptor

  2005cited by this paper
• PRT1 of Arabidopsis Is a Ubiquitin Protein Ligase of the Plant N-End Rule Pathway with Specificity for Aromatic Amino-Terminal Residues1

  2003cited by this paper
• Gravity-regulated differential auxin transport from columella to lateral root cap cells

  2003cited by this paper
• GATEWAY vectors for Agrobacterium-mediated plant transformation.

  2002cited by this paper
• Roles and activities of Aux/IAA proteins in Arabidopsis.

  2001cited by this paper
• Rapid Degradation of Auxin/Indoleacetic Acid Proteins Requires Conserved Amino Acids of Domain II and Is Proteasome Dependent

  2001cited by this paper
• IAA17/AXR3: Biochemical Insight into an Auxin Mutant Phenotype

  2001influential reference
• A Gain-of-Function Mutation in IAA28 Suppresses Lateral Root Development

  2001cited by this paper
• Auxin regulates SCFTIR1-dependent degradation of AUX/IAA proteins

  2001influential reference
• The structure of the chromophore within DsRed, a red fluorescent protein from coral.

  2000cited by this paper
• Degradation of Aux/IAA proteins is essential for normal auxin signalling.

  2000influential reference
• Ubiquitin fusion technique and its descendants.

  2000cited by this paper
• Control of auxin-regulated root development by the Arabidopsis thaliana SHY2/IAA3 gene.

  1999cited by this paper
• Changes in auxin response from mutations in an AUX/IAA gene.

  1998cited by this paper
• Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.

  1998cited by this paper
• PRT1 of Arabidopsis thaliana encodes a component of the plant N-end rule pathway.

  1998influential reference
• Mutations in the AXR3 gene of Arabidopsis result in altered auxin response including ectopic expression from the SAUR-AC1 promoter.

  1996cited by this paper
• Early auxin-induced genes encode short-lived nuclear proteins.

  1994influential reference
• In vivo half-life of a protein is a function of its amino-terminal residue.

  1986cited by this paper

• Unresolved roles of Aux/IAA proteins in auxin responses

  2025cites this paper
• Genotype-by-environment interactions shape ubiquitin-proteasome system activity

  2025cites this paper
• Evidence for Functionality of Transmembrane Domains of Predicted Non-Canonical Plant Phosphotransmitters

  2025cites this paper
• An improved toolkit of gateway- and gibson assembly-compatible vectors for protoplast transfection and agrobacterium-mediated plant transformation

  2025cites this paper
• HYPK controls stability and catalytic activity of the N-terminal acetyltransferase A in Arabidopsis thaliana.

  2024cites this paper
• A dynamic ubiquitination balance of cell proliferation and endoreduplication regulators determines plant organ size

  2024cites this paper
• Auxin co-receptor IAA17/AXR3 controls cell elongation in Arabidopsis thaliana root solely by modulation of nuclear auxin pathway.

  2024cites this paper
• Auxin coreceptor IAA17/AXR3 controls cell elongation in Arabidopsis thaliana root by modulation of auxin and gibberellin perception

  2023cites this paper
• An Improved Toolkit of Gateway- and Gibson Assembly-Compatible Vectors for Protoplast Transfection and Agrobacterium-Mediated Plant Transformation

  2023cites this paper
• Relative Protein Lifetime Measurement in Plants Using Tandem Fluorescent Protein Timers.

  2023cites this paper
• Cotranslational N-degron masking by acetylation promotes proteome stability in plants

  2022cites this paper
• High-Throughput Analysis of Protein Turnover with Tandem Fluorescent Protein Timers.

  2022cites this paper
• Indirect Methods To Measure Unfolded Proteins In Living Cells Using Fluorescent Proteins.

  2022cites this paper
• A Yeast-Based Functional Assay to Study Plant N-Degron – N-Recognin Interactions

  2022cites this paper
• HYPK promotes the activity of the Nα-acetyltransferase A complex to determine proteostasis of nonAc-X2/N-degron–containing proteins

  2022cites this paper
• Plant proteostasis: a proven and promising target for crop improvement.

  2022cites this paper
• Cotranslational N-degron masking by acetylation promotes proteome stability in plants

  2022cites this paper
• Current and future advances in fluorescence-based visualization of plant cell wall components and cell wall biosynthetic machineries

  2021cites this paper
• Substrate-specific effects of natural genetic variation on proteasome activity

  2021cites this paper
• Post-translational modifications regulate the activity of the growth restricting protease DA1.

  2021cites this paper
• The many facets of protein ubiquitination and degradation in plant root iron deficiency responses.

  2020cites this paper
• Strategies to investigate protein turnover with fluorescent protein reporters in eukaryotic organisms

  2020cites this paper
• Enzymes as Parts in Need of Replacement - and How to Extend Their Working Life.

  2020cites this paper
• Synthetic biology of hypoxia

  2020cites this paper
• The plant N-degron pathways of ubiquitin-mediated proteolysis.

  2019cites this paper
• Differential N-end Rule Degradation of RIN4/NOI Fragments Generated by the AvrRpt2 Effector Protease1[OPEN]

  2019influential citation
• N-Degron Pathways in Plastids.

  2019cites this paper
• Timing Is Everything: Tandem Fluorescent Timers Expand Our Understanding of Protein Longevity

  2019cites this paper