Wolfgang Hoehenwarter

Comparative analysis of phytohormone-responsive phosphoproteins in Arabidopsis thaliana using TiO2-phosphopeptide enrichment and mass accuracy precursor alignment

Protein phosphorylation/dephosphorylation is a central post-translational modification in plant hormone signaling, but little is known about its extent and function. Although pertinent protein kinases and phosphatases have been predicted and identified for a variety of hormone responses, classical biochemical approaches have so far revealed only a few candidate proteins and even fewer phosphorylation sites. Here we performed a global quantitative analysis of the Arabidopsis phosphoproteome in response to a time course of treatments with various plant hormones using phosphopeptide enrichment and subsequent mass accuracy precursor alignment (MAPA). The use of three time points, 1, 3 and 6 h, in combination with five phytohormone treatments, abscisic acid (ABA), indole-3-acetic acid (IAA), gibberellic acid (GA), jasmonic acid (JA) and kinetin, resulted in 324,000 precursor ions from 54 LC-Orbitrap-MS analyses quantified and aligned in a data matrix with the dimension of 6000 x 54 using the ProtMax algorithm. To dissect the phytohormone responses, multivariate principal/independent components analysis was performed. In total, 152 phosphopeptides were identified as differentially regulated; these phosphopeptides are involved in a wide variety of signaling pathways. New phosphorylation sites were identified for ABA response element binding factors that showed a specific increase in response to ABA. New phosphorylation sites were also found for RLKs and auxin transporters. We found that different hormones regulate distinct amino acid residues of members of the same protein families. In contrast, tyrosine phosphorylation of the G alpha subunit appeared to be a common response for multiple hormones, demonstrating global cross-talk among hormone signaling pathways.

Identification of Novel in vivo MAP Kinase Substrates in Arabidopsis thaliana Through Use of Tandem Metal Oxide Affinity Chromatography

Mitogen-activated protein kinase (MPK) cascades are important for eukaryotic signal transduction. They convert extracellular stimuli (e.g. some hormones, growth factors, cytokines, microbe- or damage-associated molecular patterns) into intracellular responses while at the same time amplifying the transmitting signal. By doing so, they ensure proper performance, and eventually survival, of a given organism, for example in times of stress. MPK cascades function via reversible phosphorylation of cascade components MEKKs, MEKs, and MPKs. In plants the identity of most MPK substrates remained elusive until now. Here, we provide a robust and powerful approach to identify and quantify, with high selectivity, site-specific phosphorylation of MPK substrate candidates in the model plant Arabidopsis thaliana. Our approach represents a two-step chromatography combining phosphoprotein enrichment using Al(OH)3-based metal oxide affinity chromatography, tryptic digest of enriched phosphoproteins, and TiO2-based metal oxide affinity chromatography to enrich phosphopeptides from complex protein samples. When applied to transgenic conditional gain-of-function Arabidopsis plants supporting in planta activation of MPKs, the approach allows direct measurement and quantification ex vivo of site-specific phosphorylation of several reported and many yet unknown putative MPK substrates in just a single experiment.

A rapid approach for phenotype-screening and database independent detection of cSNP/protein polymorphism using mass accuracy precursor alignment

The dynamics of a proteome can only be addressed with large‐scale, high‐throughput methods. To cope with the inherent complexity, techniques based on targeted quantification using proteotypic peptides are arising. This is an essential systems biology approach; however, for the exploratory discovery of unexpected markers, nontargeted detection of proteins, and protein modifications is indispensable. We present a rapid label‐free shotgun proteomics approach that extracts relevant phenotype‐specific peptide product ion spectra in an automated workflow without prior identification. These product ion spectra are subsequently sequenced with database search and de novo prediction algorithms. We analyzed six potato tuber cultivars grown on three plots of two geographically separated fields in Germany. For data mining about 1.5 million spectra from 107 analyses were aligned and statistically examined in approximately 1 day. Several cultivar‐specific protein markers were detected. Based on de novo‐sequencing a dominant protein polymorphism not detectable in the available EST‐databases was assigned exclusively to a specific potato cultivar. The approach is applicable to organisms with unsequenced or incomplete genomes and to the automated extraction of relevant mass spectra that potentially cannot be identified by genome/EST‐based search algorithms.

Eye lens proteomics

The eye lens is a fascinating organ as it is in essence living transparent matter. Lenticular transparency is achieved through the peculiarities of lens morphology, a semi-apoptotic process where cells elongate and loose their organelles and the precise molecular arrangement of the bulk of soluble lenticular proteins, the crystallins. The 16 crystallins ubiquitous in mammals and their modifications have been extensively characterized by 2-DE, liquid chromatography, mass spectrometry and other protein analysis techniques. The various solubility dependant fractions as well as subproteomes of lenticular morphological sections have also been explored in detail. Extensive post translational modification of the crystallins is encountered throughout the lens as a result of ageing and disease resulting in a vast number of protein species. Proteomics methodology is therefore ideal to further comprehensive understanding of this organ and the factors involved in cataractogenesis.

Spectral counting robust on high mass accuracy mass spectrometers

Mass spectrometry is central to shotgun proteomics, an application that seeks to quantify as much of the total protein complement of a biological sample as possible. The high mass accuracy, resolution, capacity and scan rate of modern mass spectrometers have greatly facilitated this endeavor. The sum of MS to MS/MS transitions in tandem mass spectrometry, the spectral count (SC), of a peptide has been shown to be a reliable estimate of its relative abundance. However, when using SCs, optimal MS configurations are crucial in order to maximize the number of low abundant proteins quantified while keeping the estimates for the highly abundant proteins within the linear dynamic range.In this study, LC/MS/MS analysis was performed using an LTQ-OrbiTrap on a sample containing many highly abundant proteins. Tuning the LTQ-OrbiTrap mass spectrometer to minimize redundant MS/MS acquisition and to maximize resolution of the proteome by accurately measured m/z ratios resulted in an appreciable increase in quantified low abundant proteins. An exclusion duration of 90 s and an exclusion width of 10 ppm were found best of those tested. The spectral count of individual proteins was found to be highly reproducible and protein abundance ratios were not affected by the different settings that were applied. We conclude that on a high mass accuracy instrument spectral counting is a robust measure of protein abundance even for samples containing many highly abundant proteins and that tuning dynamic exclusion parameters appreciably improves the number of proteins that can be reliably quantified.

ProMEX - a mass spectral reference database for plant proteomics

The ProMEX database is one of the main collection of annotated tryptic peptides in plant proteomics. The main objective of the ProMEX database is to provide experimental MS/MS-based information for cell type-specific or sub-cellular proteomes in Arabidopsis thaliana, Medicago truncatula, Chlamydomonas reinhardtii, Lotus japonicus, Lotus corniculatus, Phaseolus vulgaris, Lycopersicon esculentum, Solanum tuberosum, Nicotiana tabacum, Glycine max, Zea mays, Bradyrhizobium japonicum, and Sinorhizobium meliloti. Direct links at the protein level to the most relevant databases are present in ProMEX. Furthermore, the spectral sequence information are linked to their respective pathways and can be viewed in pathway maps.

Proteome and metabolome profiling of cytokinin action in Arabidopsis identifying both distinct and similar responses to cytokinin down- and up-regulation

In plants, numerous developmental processes are controlled by cytokinin (CK) levels and their ratios to levels of other hormones. While molecular mechanisms underlying the regulatory roles of CKs have been intensely researched, proteomic and metabolomic responses to CK deficiency are unknown. Transgenic Arabidopsis seedlings carrying inducible barley cytokinin oxidase/dehydrogenase (CaMV35S>GR>HvCKX2) and agrobacterial isopentenyl transferase (CaMV35S>GR>ipt) constructs were profiled to elucidate proteome- and metabolome-wide responses to down- and up-regulation of CK levels, respectively. Proteome profiling identified >1100 proteins, 155 of which responded to HvCKX2 and/or ipt activation, mostly involved in growth, development, and/or hormone and light signalling. The metabolome profiling covered 79 metabolites, 33 of which responded to HvCKX2 and/or ipt activation, mostly amino acids, carbohydrates, and organic acids. Comparison of the data sets obtained from activated CaMV35S>GR>HvCKX2 and CaMV35S>GR>ipt plants revealed unexpectedly extensive overlaps. Integration of the proteomic and metabolomic data sets revealed: (i) novel components of molecular circuits involved in CK action (e.g. ribosomal proteins); (ii) previously unrecognized links to redox regulation and stress hormone signalling networks; and (iii) CK content markers. The striking overlaps in profiles observed in CK-deficient and CK-overproducing seedlings might explain surprising previously reported similarities between plants with down- and up-regulated CK levels.

Multiomics of tomato glandular trichomes reveals distinct features of central carbon metabolism supporting high productivity of specialized metabolites

Multi-omics and stable isotope labeling applied to tomato glandular trichomes reveal unique features for the supply of energy, reducing power and carbon in these metabolic cell factories. Glandular trichomes are metabolic cell factories with the capacity to produce large quantities of secondary metabolites. Little is known about the connection between central carbon metabolism and metabolic productivity for secondary metabolites in glandular trichomes. To address this gap in our knowledge, we performed comparative metabolomics, transcriptomics, proteomics, and 13C-labeling of type VI glandular trichomes and leaves from a cultivated (Solanum lycopersicum LA4024) and a wild (Solanum habrochaites LA1777) tomato accession. Specific features of glandular trichomes that drive the formation of secondary metabolites could be identified. Tomato type VI trichomes are photosynthetic but acquire their carbon essentially from leaf sucrose. The energy and reducing power from photosynthesis are used to support the biosynthesis of secondary metabolites, while the comparatively reduced Calvin-Benson-Bassham cycle activity may be involved in recycling metabolic CO2. Glandular trichomes cope with oxidative stress by producing high levels of polyunsaturated fatty acids, oxylipins, and glutathione. Finally, distinct mechanisms are present in glandular trichomes to increase the supply of precursors for the isoprenoid pathways. Particularly, the citrate-malate shuttle supplies cytosolic acetyl-CoA and plastidic glycolysis and malic enzyme support the formation of plastidic pyruvate. A model is proposed on how glandular trichomes achieve high metabolic productivity.

Comparative expression profiling reveals a role of the root apoplast in local phosphate response

BackgroundPlant adaptation to limited phosphate availability comprises a wide range of responses to conserve and remobilize internal phosphate sources and to enhance phosphate acquisition. Vigorous restructuring of root system architecture provides a developmental strategy for topsoil exploration and phosphate scavenging. Changes in external phosphate availability are locally sensed at root tips and adjust root growth by modulating cell expansion and cell division. The functionally interacting Arabidopsis genes, LOW PHOSPHATE RESPONSE 1 and 2 (LPR1/LPR2) and PHOSPHATE DEFICIENCY RESPONSE 2 (PDR2), are key components of root phosphate sensing. We recently demonstrated that the LOW PHOSPHATE RESPONSE 1 - PHOSPHATE DEFICIENCY RESPONSE 2 (LPR1-PDR2) module mediates apoplastic deposition of ferric iron (Fe3+) in the growing root tip during phosphate limitation. Iron deposition coincides with sites of reactive oxygen species generation and triggers cell wall thickening and callose accumulation, which interfere with cell-to-cell communication and inhibit root growth.ResultsWe took advantage of the opposite phosphate-conditional root phenotype of the phosphate deficiency response 2 mutant (hypersensitive) and low phosphate response 1 and 2 double mutant (insensitive) to investigate the phosphate dependent regulation of gene and protein expression in roots using genome-wide transcriptome and proteome analysis. We observed an overrepresentation of genes and proteins that are involved in the regulation of iron homeostasis, cell wall remodeling and reactive oxygen species formation, and we highlight a number of candidate genes with a potential function in root adaptation to limited phosphate availability. Our experiments reveal that FERRIC REDUCTASE DEFECTIVE 3 mediated, apoplastic iron redistribution, but not intracellular iron uptake and iron storage, triggers phosphate-dependent root growth modulation. We further highlight expressional changes of several cell wall-modifying enzymes and provide evidence for adjustment of the pectin network at sites of iron accumulation in the root.ConclusionOur study reveals new aspects of the elaborate interplay between phosphate starvation responses and changes in iron homeostasis. The results emphasize the importance of apoplastic iron redistribution to mediate phosphate-dependent root growth adjustment and suggest an important role for citrate in phosphate-dependent apoplastic iron transport. We further demonstrate that root growth modulation correlates with an altered expression of cell wall modifying enzymes and changes in the pectin network of the phosphate-deprived root tip, supporting the hypothesis that pectins are involved in iron binding and/or phosphate mobilization.

Targeted Proteomics Analysis of Protein Degradation in Plant Signaling on an LTQ-Orbitrap Mass Spectrometer

Targeted proteomics has become increasingly popular recently because of its ability to precisely quantify selected proteins in complex cellular backgrounds. Here, we demonstrated the utility of an LTQ-Orbitrap Velos Pro mass spectrometer in targeted parallel reaction monitoring (PRM) despite its unconventional dual ion trap configuration. We evaluated absolute specificity (>99%) and sensitivity (100 amol on column in 1 μg of total cellular extract) using full and mass range scans as survey scans together with data-dependent (DDA) and targeted MS/MS acquisition. The instrument duty cycle was a critical parameter limiting sensitivity, necessitating peptide retention time scheduling. We assessed synthetic peptide and recombinant peptide standards to predict or experimentally determine target peptide retention times. We applied optimized PRM to protein degradation in signaling regulation, an area that is receiving increased attention in plant physiology. We quantified relative abundance of selected proteins in plants that are mutant for enzymatic components of the N-end rule degradation (NERD) pathway such as the two tRNA-arginyl-transferases ATE1 and ATE2 and the two E3 ubiquitin ligases PROTEOLYSIS1 and 6. We found a number of upregulated proteins, which might represent degradation targets. We also targeted FLAGELLIN SENSITIVE2 (FLS2), a pattern recognition receptor responsible for pathogen sensing, in ubiquitin ligase mutants to assay the attenuation of plant immunity by degradation of the receptor.