Capsaicin, produced by diverse Capsicum species, is among the world’s most popular spices and of ... more Capsaicin, produced by diverse Capsicum species, is among the world’s most popular spices and of considerable pharmaceutical relevance. Although the capsaicinoid biosynthetic pathway has been investigated for decades, several biosynthetic steps have remained partly hypothetical. Genetic evidence suggested that the decisive capsaicin synthase is encoded by the Pun1 locus. Yet, the genetic evidence of the Pun1 locus was never corroborated by functionally active capsaicin synthase that presumably catalyzes an amide bond formation between trans 8-methyl-6-nonenoyl-CoA derived from branched-chain amino acid biosynthesis and vanilloylamine derived from the phenylpropanoid pathway. In this report, we demonstrate the enzymatic activity of a recombinant capsaicin synthase encoded by Pun1, functionally expressed in Escherichia coli, and provide information on its substrate specificity and catalytic properties. Recombinant capsaicin synthase is specific for selected aliphatic CoA-esters and hi...
Endocytosis of metals in plants is a growing field of study involving metal uptake from the rhizo... more Endocytosis of metals in plants is a growing field of study involving metal uptake from the rhizosphere. Uranium, which is naturally and artificially released into the rhizosphere, is known to be taken up by certain species of plant, such as Nicotiana tabacum, and we hypothesize that endocytosis contributes to the uptake of uranium in tobacco. The endocytic uptake of uranium was investigated in tobacco BY-2 cells using an optimized setup of culture in phosphate-deficient medium. A combination of methods in biochemistry, microscopy and spectroscopy, supplemented by proteomics, were used to study the interaction of uranium and the plant cell. We found that under environmentally relevant uranium concentrations, endocytosis remained active and contributed to 14% of the total uranium bioassociation. Proteomics analyses revealed that uranium induced a change in expression of the clathrin heavy chain variant, signifying a shift in the type of endocytosis taking place. However, the rate of endocytosis remained largely unaltered. Electron microscopy and energy-dispersive X-ray spectroscopy showed an adsorption of uranium to cell surfaces and deposition in vacuoles. Our results demonstrate that endocytosis constitutes a considerable proportion of uranium uptake in BY-2 cells, and that endocytosed uranium is likely targeted to the vacuole for sequestration, providing a physiologically safer route for the plant than uranium transported through the cytosol.
For centuries, crop plants have represented the basis of the daily human diet. Among them, cereal... more For centuries, crop plants have represented the basis of the daily human diet. Among them, cereals and legumes, accumulating oils, proteins, and carbohydrates in their seeds, distinctly dominate modern agriculture, thus play an essential role in food industry and fuel production. Therefore, seeds of crop plants are intensively studied by food chemists, biologists, biochemists, and nutritional physiologists. Accordingly, seed development and germination as well as age- and stress-related alterations in seed vigor, longevity, nutritional value, and safety can be addressed by a broad panel of analytical, biochemical, and physiological methods. Currently, functional genomics is one of the most powerful tools, giving direct access to characteristic metabolic changes accompanying plant development, senescence, and response to biotic or abiotic stress. Among individual post-genomic methodological platforms, proteomics represents one of the most effective ones, giving access to cellular met...
The interaction of tobacco (<em>Nicotiana tabacum</em>) BY-2 cells with uranyl(VI) ni... more The interaction of tobacco (<em>Nicotiana tabacum</em>) BY-2 cells with uranyl(VI) nitrate in phosphate-deficient medium was investigated. The hypothesis was that endocytosis is a means of uranium uptake in these cells. Analysis was in the form of physiological studies (growth and viability), electron microscopy, proteomics and biochemical studies.
Despite its central role as the ark of genetic information and gene expression the plant nucleus ... more Despite its central role as the ark of genetic information and gene expression the plant nucleus is surprisingly understudied. We isolated nuclei from the Arabidopsis thaliana dark grown cell culture left untreated and treated with flg22 and nlp20, two elicitors of pattern triggered immunity (PTI) in plants, respectively. An liquid chromatography mass spectrometry (LC–MS) based discovery proteomics approach was used to measure the nuclear proteome fractions. An enrichment score based on the relative abundance of cytoplasmic, mitochondrial and Golgi markers in the nuclear protein fraction allowed us to curate the nuclear proteome producing high quality catalogs of around 3,000 nuclear proteins under untreated and both PTI conditions. The measurements also covered low abundant proteins including more than 100 transcription factors and transcriptional co-activators. We disclose a list of several hundred potentially dual targeted proteins including proteins not yet found before for furt...
Fluctuating bioavailability of inorganic phosphate (Pi), often caused by complex Pi-metal interac... more Fluctuating bioavailability of inorganic phosphate (Pi), often caused by complex Pi-metal interactions, guide root tip growth and root system architecture for maximizing the foraged soil volume. Two interacting genes in Arabidopsis thaliana, PDR2 (P5-type ATPase) and LPR1 (multicopper oxidase), are central to external Pi monitoring by root tips, which is modified by iron (Fe) co-occurrence. Upon Pi deficiency, the PDR2-LPR1 module facilitates cell type-specific Fe accumulation and cell wall modifications in root meristems, inhibiting intercellular communication and thus root growth. LPR1 executes local Pi sensing, whereas PDR2 restricts LPR1 function. We show that native LPR1 displays specific ferroxidase activity and requires a conserved acidic triad motif for high-affinity Fe2+ binding and root growth inhibition under limiting Pi. Our data indicate that substrate availability tunes LPR1 function and implicate PDR2 in maintaining Fe homeostasis. LPR1 represents the prototype of an ...
Fungal unspecific peroxygenases (UPOs) are biocatalysts of outstanding interest. Providing access... more Fungal unspecific peroxygenases (UPOs) are biocatalysts of outstanding interest. Providing access to novel UPOs using a modular secretion system was the central goal of this work. UPOs represent an enzyme class, catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and solely rely on hydrogen peroxide as the oxygen source. Fungal peroxygenases are widespread throughout the fungal kingdom and hence a huge variety of UPO gene sequences is available. However, the heterologous production of UPOs in a fast-growing organism suitable for high throughput screening has only succeeded once—enabled by an intensive directed evolution campaign. Here, we developed and applied a modular Golden Gate-based secretion system, allowing the first yeast production of four active UPOs, their one-step purification and application in an enantioselective conversion on a preparative scale. The G...
ABSTRACTCullin RING-type E3 ubiquitin ligases SCFTIR1/AFB1-5and their ubiquitylation targets, AUX... more ABSTRACTCullin RING-type E3 ubiquitin ligases SCFTIR1/AFB1-5and their ubiquitylation targets, AUX/IAAs, sense auxin concentrations in the nucleus. TIR1 binds a surface-exposed degron in AUX/IAAs promoting their ubiquitylation and rapid auxin-regulated proteasomal degradation. Here, we resolved TIR1·auxin·IAA7 and TIR1·auxin·IAA12 complex topology, and show that flexible intrinsically disordered regions (IDRs) in the degron’s vicinity, cooperatively position AUX/IAAs on TIR1. The AUX/IAA PB1 interaction domain also assists in non-native contacts, affecting AUX/IAA dynamic interaction states. Our results establish a role for IDRs in modulating auxin receptor assemblies. By securing AUX/IAAs on two opposite surfaces of TIR1, IDR diversity supports locally tailored positioning for targeted ubiquitylation, and might provide conformational flexibility for adopting a multiplicity of functional states. We postulate IDRs in distinct members of the AUX/IAA family to be an adaptive signature f...
Summary Ubiquitination is mediated by an enzymatic cascade that results in the modification of su... more Summary Ubiquitination is mediated by an enzymatic cascade that results in the modification of substrate proteins, redefining their fate. This post‐translational modification is involved in most cellular processes, yet its analysis faces manifold obstacles due to its complex and ubiquitous nature. Reconstitution of the ubiquitination cascade in bacterial systems circumvents several of these problems and was shown to faithfully recapitulate the process. Here, we present UbiGate − a synthetic biology toolbox, together with an inducible bacterial expression system – to enable the straightforward reconstitution of the ubiquitination cascades of different organisms in Escherichia coli by ‘Golden Gate’ cloning. This inclusive toolbox uses a hierarchical modular cloning system to assemble complex DNA molecules encoding the multiple genetic elements of the ubiquitination cascade in a predefined order, to generate polycistronic operons for expression. We demonstrate the efficiency of UbiGate...
Due to low culturing costs and high seed protein contents, legumes represent the main global sour... more Due to low culturing costs and high seed protein contents, legumes represent the main global source of food protein. Pea (Pisum sativum L.) is one of the major legume crops, impacting both animal feed and human nutrition. Therefore, the quality of pea seeds needs to be ensured in the context of sustainable crop production and nutritional efficiency. Apparently, changes in seed protein patterns might directly affect both of these aspects. Thus, here, we address the pea seed proteome in detail and provide, to the best of our knowledge, the most comprehensive annotation of the functions and intracellular localization of pea seed proteins. To address possible intercultivar differences, we compared seed proteomes of yellow- and green-seeded pea cultivars in a comprehensive case study. The analysis revealed totally 1938 and 1989 nonredundant proteins, respectively. Only 35 and 44 proteins, respectively, could be additionally identified after protamine sulfate precipitation (PSP), potentia...
Auxin is a small molecule morphogen that bridges SCF(TIR1/AFB)-AUX/IAA co-receptor interactions l... more Auxin is a small molecule morphogen that bridges SCF(TIR1/AFB)-AUX/IAA co-receptor interactions leading to ubiquitylation and proteasome-dependent degradation of AUX/IAA transcriptional repressors. Here, we systematically dissect auxin sensing by SCF(TIR1)-IAA6 and SCF(TIR1)-IAA19 co-receptor complexes, and assess IAA6/IAA19 ubiquitylation in vitro and IAA6/IAA19 degradation in vivo. We show that TIR1-IAA19 and TIR1-IAA6 have distinct auxin affinities that correlate with ubiquitylation and turnover dynamics of the AUX/IAA. We establish a system to track AUX/IAA ubiquitylation in IAA6 and IAA19 in vitro and show that it occurs in flexible hotspots in degron-flanking regions adorned with specific Lys residues. We propose that this signature is exploited during auxin-mediated SCF(TIR1)-AUX/IAA interactions. We present evidence for an evolving AUX/IAA repertoire, typified by the IAA6/IAA19 ohnologues, that discriminates the range of auxin concentrations found in plants. We postulate tha...
Crosstalk between post-translational modifications such as ubiquitination and phosphorylation pla... more Crosstalk between post-translational modifications such as ubiquitination and phosphorylation play key roles in controlling the duration and intensity of signalling events to ensure cellular homeostasis. However, the molecular mechanisms underlying the regulation of negative feedback loops remain poorly understood. Here we uncover a pathway in Arabidopsis thaliana by which a negative feedback loop involving the E3 ubiquitin ligase PUB22 that dampens the immune response is triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3), best known for its function in the activation of signalling. PUB22's stability is controlled by MPK3-mediated phosphorylation of residues localized in and adjacent to the E2 docking domain. We show that phosphorylation is critical for stabilization by inhibiting PUB22 oligomerization and thus autoubiquitination. The activity switch allows PUB22 to dampen the immune response. This regulatory mechanism also suggests that autoubiquitination, which is inherent ...
We evaluated the state of label-free discovery proteomics focusing especially on technological co... more We evaluated the state of label-free discovery proteomics focusing especially on technological contributions and contributions of naturally occurring differences in protein abundance to the intersample variability in protein abundance estimates in this highly peptide-centric technology. First, the performance of popular quantitative proteomics software, Proteome Discoverer, Scaffold, MaxQuant, and Progenesis QIP, was benchmarked using their default parameters and some modified settings. Beyond this, the intersample variability in protein abundance estimates was decomposed into variability introduced by the entire technology itself and variable protein amounts inherent to individual plants of the Arabidopsis thaliana Col-0 accession. The technical component was considerably higher than the biological intersample variability, suggesting an effect on the degree and validity of reported biological changes in protein abundance. Surprisingly, the biological variability, protein abundance estimates, and protein fold changes were recorded differently by the software used to quantify the proteins, warranting caution in the comparison of discovery proteomics results. As expected, ∼99% of the proteome was invariant in the isogenic plants in the absence of environmental factors; however, few proteins showed substantial quantitative variability. This naturally occurring variation between individual organisms can have an impact on the causality of reported protein fold changes.
Phenotypes on-demand generated by controlling activation and accumulation of proteins of interest... more Phenotypes on-demand generated by controlling activation and accumulation of proteins of interest are invaluable tools to analyse and engineer biological processes. While temperature-sensitive alleles are frequently used as conditional mutants in microorganisms, they are usually difficult to identify in multicellular species. Here we present a versatile and transferable, genetically stable system based on a low-temperature-controlled N-terminal degradation signal (lt-degron) that allows reversible and switch-like tuning of protein levels under physiological conditions in vivo. Thereby, developmental effects can be triggered and phenotypes on demand generated. The lt-degron was established to produce conditional and cell-type-specific phenotypes and is generally applicable in a wide range of organisms, from eukaryotic microorganisms to plants and poikilothermic animals. We have successfully applied this system to control the abundance and function of transcription factors and differe...
Capsaicin, produced by diverse Capsicum species, is among the world’s most popular spices and of ... more Capsaicin, produced by diverse Capsicum species, is among the world’s most popular spices and of considerable pharmaceutical relevance. Although the capsaicinoid biosynthetic pathway has been investigated for decades, several biosynthetic steps have remained partly hypothetical. Genetic evidence suggested that the decisive capsaicin synthase is encoded by the Pun1 locus. Yet, the genetic evidence of the Pun1 locus was never corroborated by functionally active capsaicin synthase that presumably catalyzes an amide bond formation between trans 8-methyl-6-nonenoyl-CoA derived from branched-chain amino acid biosynthesis and vanilloylamine derived from the phenylpropanoid pathway. In this report, we demonstrate the enzymatic activity of a recombinant capsaicin synthase encoded by Pun1, functionally expressed in Escherichia coli, and provide information on its substrate specificity and catalytic properties. Recombinant capsaicin synthase is specific for selected aliphatic CoA-esters and hi...
Endocytosis of metals in plants is a growing field of study involving metal uptake from the rhizo... more Endocytosis of metals in plants is a growing field of study involving metal uptake from the rhizosphere. Uranium, which is naturally and artificially released into the rhizosphere, is known to be taken up by certain species of plant, such as Nicotiana tabacum, and we hypothesize that endocytosis contributes to the uptake of uranium in tobacco. The endocytic uptake of uranium was investigated in tobacco BY-2 cells using an optimized setup of culture in phosphate-deficient medium. A combination of methods in biochemistry, microscopy and spectroscopy, supplemented by proteomics, were used to study the interaction of uranium and the plant cell. We found that under environmentally relevant uranium concentrations, endocytosis remained active and contributed to 14% of the total uranium bioassociation. Proteomics analyses revealed that uranium induced a change in expression of the clathrin heavy chain variant, signifying a shift in the type of endocytosis taking place. However, the rate of endocytosis remained largely unaltered. Electron microscopy and energy-dispersive X-ray spectroscopy showed an adsorption of uranium to cell surfaces and deposition in vacuoles. Our results demonstrate that endocytosis constitutes a considerable proportion of uranium uptake in BY-2 cells, and that endocytosed uranium is likely targeted to the vacuole for sequestration, providing a physiologically safer route for the plant than uranium transported through the cytosol.
For centuries, crop plants have represented the basis of the daily human diet. Among them, cereal... more For centuries, crop plants have represented the basis of the daily human diet. Among them, cereals and legumes, accumulating oils, proteins, and carbohydrates in their seeds, distinctly dominate modern agriculture, thus play an essential role in food industry and fuel production. Therefore, seeds of crop plants are intensively studied by food chemists, biologists, biochemists, and nutritional physiologists. Accordingly, seed development and germination as well as age- and stress-related alterations in seed vigor, longevity, nutritional value, and safety can be addressed by a broad panel of analytical, biochemical, and physiological methods. Currently, functional genomics is one of the most powerful tools, giving direct access to characteristic metabolic changes accompanying plant development, senescence, and response to biotic or abiotic stress. Among individual post-genomic methodological platforms, proteomics represents one of the most effective ones, giving access to cellular met...
The interaction of tobacco (<em>Nicotiana tabacum</em>) BY-2 cells with uranyl(VI) ni... more The interaction of tobacco (<em>Nicotiana tabacum</em>) BY-2 cells with uranyl(VI) nitrate in phosphate-deficient medium was investigated. The hypothesis was that endocytosis is a means of uranium uptake in these cells. Analysis was in the form of physiological studies (growth and viability), electron microscopy, proteomics and biochemical studies.
Despite its central role as the ark of genetic information and gene expression the plant nucleus ... more Despite its central role as the ark of genetic information and gene expression the plant nucleus is surprisingly understudied. We isolated nuclei from the Arabidopsis thaliana dark grown cell culture left untreated and treated with flg22 and nlp20, two elicitors of pattern triggered immunity (PTI) in plants, respectively. An liquid chromatography mass spectrometry (LC–MS) based discovery proteomics approach was used to measure the nuclear proteome fractions. An enrichment score based on the relative abundance of cytoplasmic, mitochondrial and Golgi markers in the nuclear protein fraction allowed us to curate the nuclear proteome producing high quality catalogs of around 3,000 nuclear proteins under untreated and both PTI conditions. The measurements also covered low abundant proteins including more than 100 transcription factors and transcriptional co-activators. We disclose a list of several hundred potentially dual targeted proteins including proteins not yet found before for furt...
Fluctuating bioavailability of inorganic phosphate (Pi), often caused by complex Pi-metal interac... more Fluctuating bioavailability of inorganic phosphate (Pi), often caused by complex Pi-metal interactions, guide root tip growth and root system architecture for maximizing the foraged soil volume. Two interacting genes in Arabidopsis thaliana, PDR2 (P5-type ATPase) and LPR1 (multicopper oxidase), are central to external Pi monitoring by root tips, which is modified by iron (Fe) co-occurrence. Upon Pi deficiency, the PDR2-LPR1 module facilitates cell type-specific Fe accumulation and cell wall modifications in root meristems, inhibiting intercellular communication and thus root growth. LPR1 executes local Pi sensing, whereas PDR2 restricts LPR1 function. We show that native LPR1 displays specific ferroxidase activity and requires a conserved acidic triad motif for high-affinity Fe2+ binding and root growth inhibition under limiting Pi. Our data indicate that substrate availability tunes LPR1 function and implicate PDR2 in maintaining Fe homeostasis. LPR1 represents the prototype of an ...
Fungal unspecific peroxygenases (UPOs) are biocatalysts of outstanding interest. Providing access... more Fungal unspecific peroxygenases (UPOs) are biocatalysts of outstanding interest. Providing access to novel UPOs using a modular secretion system was the central goal of this work. UPOs represent an enzyme class, catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and solely rely on hydrogen peroxide as the oxygen source. Fungal peroxygenases are widespread throughout the fungal kingdom and hence a huge variety of UPO gene sequences is available. However, the heterologous production of UPOs in a fast-growing organism suitable for high throughput screening has only succeeded once—enabled by an intensive directed evolution campaign. Here, we developed and applied a modular Golden Gate-based secretion system, allowing the first yeast production of four active UPOs, their one-step purification and application in an enantioselective conversion on a preparative scale. The G...
ABSTRACTCullin RING-type E3 ubiquitin ligases SCFTIR1/AFB1-5and their ubiquitylation targets, AUX... more ABSTRACTCullin RING-type E3 ubiquitin ligases SCFTIR1/AFB1-5and their ubiquitylation targets, AUX/IAAs, sense auxin concentrations in the nucleus. TIR1 binds a surface-exposed degron in AUX/IAAs promoting their ubiquitylation and rapid auxin-regulated proteasomal degradation. Here, we resolved TIR1·auxin·IAA7 and TIR1·auxin·IAA12 complex topology, and show that flexible intrinsically disordered regions (IDRs) in the degron’s vicinity, cooperatively position AUX/IAAs on TIR1. The AUX/IAA PB1 interaction domain also assists in non-native contacts, affecting AUX/IAA dynamic interaction states. Our results establish a role for IDRs in modulating auxin receptor assemblies. By securing AUX/IAAs on two opposite surfaces of TIR1, IDR diversity supports locally tailored positioning for targeted ubiquitylation, and might provide conformational flexibility for adopting a multiplicity of functional states. We postulate IDRs in distinct members of the AUX/IAA family to be an adaptive signature f...
Summary Ubiquitination is mediated by an enzymatic cascade that results in the modification of su... more Summary Ubiquitination is mediated by an enzymatic cascade that results in the modification of substrate proteins, redefining their fate. This post‐translational modification is involved in most cellular processes, yet its analysis faces manifold obstacles due to its complex and ubiquitous nature. Reconstitution of the ubiquitination cascade in bacterial systems circumvents several of these problems and was shown to faithfully recapitulate the process. Here, we present UbiGate − a synthetic biology toolbox, together with an inducible bacterial expression system – to enable the straightforward reconstitution of the ubiquitination cascades of different organisms in Escherichia coli by ‘Golden Gate’ cloning. This inclusive toolbox uses a hierarchical modular cloning system to assemble complex DNA molecules encoding the multiple genetic elements of the ubiquitination cascade in a predefined order, to generate polycistronic operons for expression. We demonstrate the efficiency of UbiGate...
Due to low culturing costs and high seed protein contents, legumes represent the main global sour... more Due to low culturing costs and high seed protein contents, legumes represent the main global source of food protein. Pea (Pisum sativum L.) is one of the major legume crops, impacting both animal feed and human nutrition. Therefore, the quality of pea seeds needs to be ensured in the context of sustainable crop production and nutritional efficiency. Apparently, changes in seed protein patterns might directly affect both of these aspects. Thus, here, we address the pea seed proteome in detail and provide, to the best of our knowledge, the most comprehensive annotation of the functions and intracellular localization of pea seed proteins. To address possible intercultivar differences, we compared seed proteomes of yellow- and green-seeded pea cultivars in a comprehensive case study. The analysis revealed totally 1938 and 1989 nonredundant proteins, respectively. Only 35 and 44 proteins, respectively, could be additionally identified after protamine sulfate precipitation (PSP), potentia...
Auxin is a small molecule morphogen that bridges SCF(TIR1/AFB)-AUX/IAA co-receptor interactions l... more Auxin is a small molecule morphogen that bridges SCF(TIR1/AFB)-AUX/IAA co-receptor interactions leading to ubiquitylation and proteasome-dependent degradation of AUX/IAA transcriptional repressors. Here, we systematically dissect auxin sensing by SCF(TIR1)-IAA6 and SCF(TIR1)-IAA19 co-receptor complexes, and assess IAA6/IAA19 ubiquitylation in vitro and IAA6/IAA19 degradation in vivo. We show that TIR1-IAA19 and TIR1-IAA6 have distinct auxin affinities that correlate with ubiquitylation and turnover dynamics of the AUX/IAA. We establish a system to track AUX/IAA ubiquitylation in IAA6 and IAA19 in vitro and show that it occurs in flexible hotspots in degron-flanking regions adorned with specific Lys residues. We propose that this signature is exploited during auxin-mediated SCF(TIR1)-AUX/IAA interactions. We present evidence for an evolving AUX/IAA repertoire, typified by the IAA6/IAA19 ohnologues, that discriminates the range of auxin concentrations found in plants. We postulate tha...
Crosstalk between post-translational modifications such as ubiquitination and phosphorylation pla... more Crosstalk between post-translational modifications such as ubiquitination and phosphorylation play key roles in controlling the duration and intensity of signalling events to ensure cellular homeostasis. However, the molecular mechanisms underlying the regulation of negative feedback loops remain poorly understood. Here we uncover a pathway in Arabidopsis thaliana by which a negative feedback loop involving the E3 ubiquitin ligase PUB22 that dampens the immune response is triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3), best known for its function in the activation of signalling. PUB22's stability is controlled by MPK3-mediated phosphorylation of residues localized in and adjacent to the E2 docking domain. We show that phosphorylation is critical for stabilization by inhibiting PUB22 oligomerization and thus autoubiquitination. The activity switch allows PUB22 to dampen the immune response. This regulatory mechanism also suggests that autoubiquitination, which is inherent ...
We evaluated the state of label-free discovery proteomics focusing especially on technological co... more We evaluated the state of label-free discovery proteomics focusing especially on technological contributions and contributions of naturally occurring differences in protein abundance to the intersample variability in protein abundance estimates in this highly peptide-centric technology. First, the performance of popular quantitative proteomics software, Proteome Discoverer, Scaffold, MaxQuant, and Progenesis QIP, was benchmarked using their default parameters and some modified settings. Beyond this, the intersample variability in protein abundance estimates was decomposed into variability introduced by the entire technology itself and variable protein amounts inherent to individual plants of the Arabidopsis thaliana Col-0 accession. The technical component was considerably higher than the biological intersample variability, suggesting an effect on the degree and validity of reported biological changes in protein abundance. Surprisingly, the biological variability, protein abundance estimates, and protein fold changes were recorded differently by the software used to quantify the proteins, warranting caution in the comparison of discovery proteomics results. As expected, ∼99% of the proteome was invariant in the isogenic plants in the absence of environmental factors; however, few proteins showed substantial quantitative variability. This naturally occurring variation between individual organisms can have an impact on the causality of reported protein fold changes.
Phenotypes on-demand generated by controlling activation and accumulation of proteins of interest... more Phenotypes on-demand generated by controlling activation and accumulation of proteins of interest are invaluable tools to analyse and engineer biological processes. While temperature-sensitive alleles are frequently used as conditional mutants in microorganisms, they are usually difficult to identify in multicellular species. Here we present a versatile and transferable, genetically stable system based on a low-temperature-controlled N-terminal degradation signal (lt-degron) that allows reversible and switch-like tuning of protein levels under physiological conditions in vivo. Thereby, developmental effects can be triggered and phenotypes on demand generated. The lt-degron was established to produce conditional and cell-type-specific phenotypes and is generally applicable in a wide range of organisms, from eukaryotic microorganisms to plants and poikilothermic animals. We have successfully applied this system to control the abundance and function of transcription factors and differe...
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