Fluorescence-activated multi-organelle mapping of subcellular plant hormone distribution.

Auxins and cytokinins are two major families of phytohormones that control most aspects of plant growth, development and plasticity. Their distribution in plants has been described, but the importance of cell- and subcellular-type specific phytohormone homeostasis remains undefined. Herein, we revealed auxin and cytokinin distribution maps showing their different organelle-specific allocations within the Arabidopsis plant cell. To do so, we have developed Fluorescence-Activated multi-Organelle Sorting (FAmOS), an innovative subcellular fractionation technique based on flow cytometric principles. FAmOS allows the simultaneous sorting of four differently labelled organelles based on their individual light scatter and fluorescence parameters while ensuring hormone metabolic stability. Our data showed different subcellular distribution of auxin and cytokinins, revealing the formation of phytohormone gradients that have been suggested by the subcellular localization of auxin and cytokinin transporters, receptors and metabolic enzymes. Both hormones showed enrichment in vacuoles, while cytokinins were also accumulated in the endoplasmic reticulum.

Is the co-option of jasmonate signalling for botanical carnivory a universal trait for all carnivorous plants?

The carnivorous plants in the order Caryophyllales co-opted jasmonate signalling from plant defence to botanical carnivory. However, carnivorous plants have at least 11 independent origins, and here we ask whether jasmonate signalling has been co-opted repeatedly in different evolutionary lineages. We experimentally wounded and fed the carnivorous plants Sarracenia purpurea (order Ericales), Cephalotus follicularis (order Oxalidales), Drosophyllum lusitanicum (order Caryophyllales), and measured electrical signals, phytohormone tissue level, and digestive enzymes activity. Coronatine was added exogenously to confirm the role of jasmonates in the induction of digestive process. Immunodetection of aspartic protease and proteomic analysis of digestive fluid was also performed. We found that prey capture induced accumulation of endogenous jasmonates only in D. lusitanicum, in accordance with increased enzyme activity after insect prey or coronatine application. In C. follicularis, the enzyme activity was constitutive while in S. purpurea was regulated by multiple factors. Several classes of digestive enzymes were identified in the digestive fluid of D. lusitanicum. Although carnivorous plants from different evolutionary lineages use the same digestive enzymes, the mechanism of their regulation differs. All investigated genera use jasmonates for their ancient role, defence, but jasmonate signalling has been co-opted for botanical carnivory only in some of them.

Pyocin-mediated antagonistic interactions in Pseudomonas spp. isolated in James Ross Island, Antarctica.

Interactions within bacterial communities are frequently mediated by the production of antimicrobial agents. Despite the increasing interest in research of new antimicrobials, studies describing antagonistic interactions among cold-adapted microorganisms are still rare. Our study assessed the antimicrobial interactions of 36 Antarctic Pseudomonas spp. and described the genetic background of these interactions in selected strains. The overall bacteriocinogeny was greater compared to mesophilic Pseudomonas non-aeruginosa species. R-type tailocins were detected on transmission electron micrographs in 16 strains (44.4%); phylogenetic analysis of the corresponding gene clusters revealed that the P. prosekii CCM 8878 tailocin was related to the Rp3 group, whereas the tailocin in Pseudomonas sp. CCM 8880 to the Rp4 group. Soluble antimicrobials were produced by eight strains (22.-2%); gene mining found pyocin L homologues in the genomes of P. prosekii CCM 8881 and CCM 8879 and pyocin S9-like homologues in P. prosekii CCM 8881 and Pseudomonas sp. CCM 8880. Analysis of secretomes confirmed the production of all S- and L-type pyocin genes. Our results suggest that bacteriocin-based inhibition plays an important role in interactions among Antarctic soil bacteria, and these native, cold-adapted microorganisms could be a promising source of new antimicrobials.

Unique organization of photosystem II supercomplexes and megacomplexes in Norway spruce.

Photosystem II (PSII) complexes are organized into large supercomplexes with variable amounts of light-harvesting proteins (Lhcb). A typical PSII supercomplex in plants is formed by four trimers of Lhcb proteins (LHCII trimers), which are bound to the PSII core dimer via monomeric antenna proteins. However, the architecture of PSII supercomplexes in Norway spruce[Picea abies (L.) Karst.] is different, most likely due to a lack of two Lhcb proteins, Lhcb6 and Lhcb3. Interestingly, the spruce PSII supercomplex shares similar structural features with its counterpart in the green alga Chlamydomonas reinhardtii [Kouril et al. (2016) New Phytol. 210, 808-814]. Here we present a single-particle electron microscopy study of isolated PSII supercomplexes from Norway spruce that revealed binding of a variable amount of LHCII trimers to the PSII core dimer at positions that have never been observed in any other plant species so far. The largest spruce PSII supercomplex, which was found to bind eight LHCII trimers, is even larger than the current largest known PSII supercomplex from C. reinhardtii. We have also shown that the spruce PSII supercomplexes can form various types of PSII megacomplexes, which were also identified in intact grana membranes. Some of these large PSII supercomplexes and megacomplexes were identified also in Pinus sylvestris, another representative of the Pinaceae family. The structural variability and complexity of LHCII organization in Pinaceae seems to be related to the absence of Lhcb6 and Lhcb3 in this family, and may be beneficial for the optimization of light-harvesting under varying environmental conditions.

Auxin Metabolome Profiling in the Arabidopsis Endoplasmic Reticulum Using an Optimised Organelle Isolation Protocol.

The endoplasmic reticulum (ER) is an extensive network of intracellular membranes. Its major functions include proteosynthesis, protein folding, post-transcriptional modification and sorting of proteins within the cell, and lipid anabolism. Moreover, several studies have suggested that it may be involved in regulating intracellular auxin homeostasis in plants by modulating its metabolism. Therefore, to study auxin metabolome in the ER, it is necessary to obtain a highly enriched (ideally, pure) ER fraction. Isolation of the ER is challenging because its biochemical properties are very similar to those of other cellular endomembranes. Most published protocols for ER isolation use density gradient ultracentrifugation, despite its suboptimal resolving power. Here we present an optimised protocol for ER isolation from Arabidopsis thaliana seedlings for the subsequent mass spectrometric determination of ER-specific auxin metabolite profiles. Auxin metabolite analysis revealed highly elevated levels of active auxin form (IAA) within the ER compared to whole plants. Moreover, samples prepared using our optimised isolation ER protocol are amenable to analysis using various "omics" technologies including analyses of both macromolecular and low molecular weight compounds from the same sample.

Jasmonate-independent regulation of digestive enzyme activity in the carnivorous butterwort Pinguicula × Tina.

Carnivorous plants within the order Caryophyllales use jasmonates, a class of phytohormone, in the regulation of digestive enzyme activities. We used the carnivorous butterwort Pinguicula × Tina from the order Lamiales to investigate whether jasmonate signaling is a universal and ubiquitous signaling pathway that exists outside the order Caryophyllales. We measured the electrical signals, enzyme activities, and phytohormone tissue levels in response to prey capture. Mass spectrometry was used to identify proteins in the digestive secretion. We identified eight enzymes in the digestive secretion, many of which were previously found in other genera of carnivorous plants. Among them, alpha-amylase is unique in carnivorous plants. Enzymatic activities increased in response to prey capture; however, the tissue content of jasmonic acid and its isoleucine conjugate remained rather low in contrast to the jasmonate response to wounding. Enzyme activities did not increase in response to the exogenous application of jasmonic acid or coronatine. Whereas similar digestive enzymes were co-opted from plant defense mechanisms among carnivorous plants, the mode of their regulation differs. The butterwort has not co-opted jasmonate signaling for the induction of enzyme activities in response to prey capture. Moreover, the presence of alpha-amylase in digestive fluid of P. × Tina, which has not been found in other genera of carnivorous plants, might indicate that non-defense-related genes have also been co-opted for carnivory.

Chemical proteomic analysis of 6-benzylaminopurine molecular partners in wheat grains.

KEY MESSAGE: An affinity-based chemical proteomic technique enabled direct identification of BAP-interacting proteins in wheat, including the well-known cytokinin-binder, cytokinin-binding protein 1. In this work, we show the development of a chemical proteomic technique for the identification of proteins binding to natural aromatic cytokinins (CKs). 6-benzylaminopurine (BAP) and documented CK-binder, wheat germ-allocated cytokinin-binding protein 1 (CBP-1), were suggested as an ideal proof-of concept affinity pair. Therefore, wheat grains were chosen as a model plant material. The BAP affinity beads were prepared by the immobilization of synthesized BAP-derived ligand to a commercial, pre-activated resin and used to isolate target proteins. The proteomic analysis of complex plant extracts is often complicated by the presence of highly abundant background proteins; in this case, the omnipresent alpha-amylase inhibitors (AAIs). To cope with this problem, we included SDS-PAGE, in-gel trypsin digestion and fraction pooling prior to shotgun analysis, which brought about an obvious drop in the signals belonging to the obstructing proteins. This was accompanied by a sharp increase in the number of identified BAP targets in comparison to a conventional in-solution digestion approach. To distinguish specific CK-binding proteins from those having a general affinity for nucleotide-like compounds, competitive pull-downs with natural nucleotides and free BAP were included in every affinity experiment. By this approach, we were able to identify a group of BAP-interacting proteins, which were subsequently found to be related to biological processes affected by CKs. Moreover, the selected affinity enrichment strategy was verified by the detection of the aforementioned CK-interacting protein, CBP-1. We propose that the developed method represents a promising tool for appealing research of as yet unknown CK molecular partners in plants.

Role of DNA Repair Factor Xeroderma Pigmentosum Protein Group C in Response to Replication Stress As Revealed by DNA Fragile Site Affinity Chromatography and Quantitative Proteomics.

Replication stress (RS) fuels genomic instability and cancer development and may contribute to aging, raising the need to identify factors involved in cellular responses to such stress. Here, we present a strategy for identification of factors affecting the maintenance of common fragile sites (CFSs), which are genomic loci that are particularly sensitive to RS and suffer from increased breakage and rearrangements in tumors. A DNA probe designed to match the high flexibility island sequence typical for the commonly expressed CFS (FRA16D) was used as specific DNA affinity bait. Proteins significantly enriched at the FRA16D fragment under normal and replication stress conditions were identified using stable isotope labeling of amino acids in cell culture-based quantitative mass spectrometry. The identified proteins interacting with the FRA16D fragment included some known CFS stabilizers, thereby validating this screening approach. Among the hits from our screen so far not implicated in CFS maintenance, we chose Xeroderma pigmentosum protein group C (XPC) for further characterization. XPC is a key factor in the DNA repair pathway known as global genomic nucleotide excision repair (GG-NER), a mechanism whose several components were enriched at the FRA16D fragment in our screen. Functional experiments revealed defective checkpoint signaling and escape of DNA replication intermediates into mitosis and the next generation of XPC-depleted cells exposed to RS. Overall, our results provide insights into an unexpected biological role of XPC in response to replication stress and document the power of proteomics-based screening strategies to elucidate mechanisms of pathophysiological significance.

Identification of Bremia lactucae and Oidium neolycopersici proteins extracted for intact spore MALDI mass spectrometric biotyping.

Several proteomic approaches were applied to identify protein markers providing typical signals during intact cell/spore (IC/IS) MALDI-TOF MS of two plant pathogens, namely Bremia lactucae (a downy mildew) and Oidium neolycopersici (a powdery mildew). First, proteins were extracted from intact spores of the microorganisms under conditions simulating their treatment prior to the mass spectrometric analysis. After a separation by electrophoresis and tryptic digestion, 198 and 140 proteins were identified in the B. lactucae and O. neolycopersici extracts, respectively. A large portion of them were found to be involved in the process of protein biosynthesis. For the first time, some proteins were assigned to characteristic signals in MS profiles of the investigated pathogens based on an agreement in the molecular mass. There were 9 and 10 proteins recognized, respectively, which could contribute significantly to the spectral patterns. These proteins were assigned tentatively to the following peaks in the MS profiles: (i) m/z 7828; 8593; 10 456; 11 312; 12 450; 12 763; 14 756 and 16 854 for B. lactucae; (ii) m/z 7709; 8895; 9504; 9952; 11 317; 14 082 and 14 839 for O. neolycopersici. We demonstrated the presence of ribosomal proteins and histones, which could be employed as markers in biotyping analyses for pathogen identification.

Structural characterization of a plant photosystem I and NAD(P)H dehydrogenase supercomplex.

Cyclic electron transport (CET) around photosystem I (PSI) plays an important role in balancing the ATP/NADPH ratio and the photoprotection of plants. The NAD(P)H dehydrogenase complex (NDH) has a key function in one of the CET pathways. Current knowledge indicates that, in order to fulfill its role in CET, the NDH complex needs to be associated with PSI; however, until now there has been no direct structural information about such a supercomplex. Here we present structural data obtained for a plant PSI-NDH supercomplex. Electron microscopy analysis revealed that in this supercomplex two copies of PSI are attached to one NDH complex. A constructed pseudo-atomic model indicates asymmetric binding of two PSI complexes to NDH and suggests that the low-abundant Lhca5 and Lhca6 subunits mediate the binding of one of the PSI complexes to NDH. On the basis of our structural data, we propose a model of electron transport in the PSI-NDH supercomplex in which the association of PSI to NDH seems to be important for efficient trapping of reduced ferredoxin by NDH.

Proteomic analysis of barley cell nuclei purified by flow sorting.

Many proteins are present in the nucleus; some are involved with its structural and functional organization, some with gene expression, and some with cell division. The plant nuclear proteome has not been well explored. Its characterization requires extraction methods which minimize both the artifactual alteration of the proteins and the extent of contamination with non-nuclear proteins. The conventional multi-step fractionation procedure is both laborious and prone to contamination. Here, we describe a single-step method based on flow sorting. The method allows the separation of G1, S and G2 phase nuclei and minimizes the risk of contamination by non-nuclear proteins. Preliminary results obtained using G1 phase cell nuclei from barley root tips indicate that flow sorting coupled with a protein/peptide separation and mass spectrometry will permit a comprehensive characterization of the plant nuclear proteome.

Exploring affinity chromatography in proteomics: A comprehensive review.

Over the past decades, the proteomics field has undergone rapid growth. Progress in mass spectrometry and bioinformatics, together with separation methods, has brought many innovative approaches to the study of the molecular biology of the cell. The potential of affinity chromatography was recognized immediately after its first application in proteomics, and since that time, it has become one of the cornerstones of many proteomic protocols. Indeed, this chromatographic technique exploiting the specific binding between two molecules has been employed for numerous purposes, from selective removal of interfering (over)abundant proteins or enrichment of scarce biomarkers in complex biological samples to mapping the post-translational modifications and protein interactions with other proteins, nucleic acids or biologically active small molecules. This review presents a comprehensive survey of this versatile analytical tool in current proteomics. To navigate the reader, the haphazard space of affinity separations is classified according to the experiment's aims and the separated molecule's nature. Different types of available ligands and experimental strategies are discussed in further detail for each of the mentioned procedures.

A miniaturized chemical proteomic approach for target profiling of clinical kinase inhibitors in tumor biopsies.

While targeted therapy based on the idea of attenuating the activity of a preselected, therapeutically relevant protein has become one of the major trends in modern cancer therapy, no truly specific targeted drug has been developed and most clinical agents have displayed a degree of polypharmacology. Therefore, the specificity of anticancer therapeutics has emerged as a highly important but severely underestimated issue. Chemical proteomics is a powerful technique combining postgenomic drug-affinity chromatography with high-end mass spectrometry analysis and bioinformatic data processing to assemble a target profile of a desired therapeutic molecule. Due to high demands on the starting material, however, chemical proteomic studies have been mostly limited to cancer cell lines. Herein, we report a down-scaling of the technique to enable the analysis of very low abundance samples, as those obtained from needle biopsies. By a systematic investigation of several important parameters in pull-downs with the multikinase inhibitor bosutinib, the standard experimental protocol was optimized to 100 µg protein input. At this level, more than 30 well-known targets were detected per single pull-down replicate with high reproducibility. Moreover, as presented by the comprehensive target profile obtained from miniaturized pull-downs with another clinical drug, dasatinib, the optimized protocol seems to be extendable to other drugs of interest. Sixty distinct human and murine targets were finally identified for bosutinib and dasatinib in chemical proteomic experiments utilizing core needle biopsy samples from xenotransplants derived from patient tumor tissue. Altogether, the developed methodology proves robust and generic and holds many promises for the field of personalized health care.

Proteome Analysis of Condensed Barley Mitotic Chromosomes.

Proteins play a major role in the three-dimensional organization of nuclear genome and its function. While histones arrange DNA into a nucleosome fiber, other proteins contribute to higher-order chromatin structures in interphase nuclei, and mitotic/meiotic chromosomes. Despite the key role of proteins in maintaining genome integrity and transferring hereditary information to daughter cells and progenies, the knowledge about their function remains fragmentary. This is particularly true for the proteins of condensed chromosomes and, in particular, chromosomes of plants. Here, we purified barley mitotic metaphase chromosomes by a flow cytometric sorting and characterized their proteins. Peptides from tryptic protein digests were fractionated either on a cation exchanger or reversed-phase microgradient system before liquid chromatography coupled to tandem mass spectrometry. Chromosomal proteins comprising almost 900 identifications were classified based on a combination of software prediction, available database localization information, sequence homology, and domain representation. A biological context evaluation indicated the presence of several groups of abundant proteins including histones, topoisomerase 2, POLYMERASE 2, condensin subunits, and many proteins with chromatin-related functions. Proteins involved in processes related to DNA replication, transcription, and repair as well as nucleolar proteins were found. We have experimentally validated the presence of FIBRILLARIN 1, one of the nucleolar proteins, on metaphase chromosomes, suggesting that plant chromosomes are coated with proteins during mitosis, similar to those of human and animals. These results improve significantly the knowledge of plant chromosomal proteins and provide a basis for their functional characterization and comparative phylogenetic analyses.

Pharmacological targeting of CDK9 in cardiac hypertrophy.

Cardiac hypertrophy allows the heart to adapt to workload, but persistent or unphysiological stimulus can result in pump failure. Cardiac hypertrophy is characterized by an increase in the size of differentiated cardiac myocytes. At the molecular level, growth of cells is linked to intensive transcription and translation. Several cyclin-dependent kinases (CDKs) have been identified as principal regulators of transcription, and among these CDK9 is directly associated with cardiac hypertrophy. CDK9 phosphorylates the C-terminal domain of RNA polymerase II and thus stimulates the elongation phase of transcription. Chronic activation of CDK9 causes not only cardiac myocyte enlargement but also confers predisposition to heart failure. Due to the long interest of molecular oncologists and medicinal chemists in CDKs as potential targets of anticancer drugs, a portfolio of small-molecule inhibitors of CDK9 is available. Recent determination of CDK9's crystal structure now allows the development of selective inhibitors and their further optimization in terms of biochemical potency and selectivity. CDK9 may therefore constitute a novel target for drugs against cardiac hypertrophy.

Novel modification of growth medium enables efficient E. coli expression and simple purification of an endotoxin-free recombinant murine hsp70 protein.

Heat shock protein 70 kDa (hsp70), a molecular chaperone involved in folding of nascent proteins, has been studied for its ability to activate innate and specific immunity. High purity hsp70 preparation is generally required for immunization experiments, because endotoxins and other immunologically active contaminants may affect immune responses independently of hsp70. We have developed a novel modification of E. coli-expression medium that enabled a simple two-step production and purification method for endotoxin-free recombinant hsp70. During Ni-NTA-based affinity purification of hsp70, a contaminating protein from host E. coli cells, L-glutamine-D-fructose-6-phosphate aminotransferase (GFAT), was identified. By testing various compounds, supplementation of growth medium with a GFAT metabolite, N-acetylglucosamine, was found to reduce GFAT expression and increase the total hsp70 yield five times. The new protocol is based on column purification of His-tagged hsp70 protein produced by E. coli with the modified medium, followed by endotoxin removal by Triton X-114 extraction. This approach yielded hsp70 with high purity and minimal endotoxin contamination, making the final product acceptable for immunization experiments. In summary, a simple modification of growth medium allowed production of recombinant mouse hsp70 in high yield and purity, thus compatible with immunological studies. This protocol may be useful for production of other His.

Identification of Plant Nuclear Proteins Based on a Combination of Flow Sorting, SDS-PAGE, and LC-MS/MS Analysis.

In the plant nucleus, the majority of cellular DNA content is stored and maintained. This makes this highly specialized organelle the major coordinator of almost all essential processes in plant cells such as transcription, DNA replication, and repair. None of these biological pathways can be fully understood without a comprehensive characterization of nuclear proteins. Nevertheless, the interest of the proteomic community in the plant nuclear proteome has been very limited so far. This is probably due to the high integrity of plant cell, presence of many interfering metabolites, and considerable endogenous proteolytic activity which make the sample preparation problematic. Hereby, we describe a novel protocol for the high-throughput plant nuclear protein identification that combines a flow cytometric sorting of formaldehyde-fixed nuclei with protein and peptide separation and their subsequent LC-MS/MS analysis.

UNcleProt (Universal Nuclear Protein database of barley): The first nuclear protein database that distinguishes proteins from different phases of the cell cycle.

Proteins are the most abundant component of the cell nucleus, where they perform a plethora of functions, including the assembly of long DNA molecules into condensed chromatin, DNA replication and repair, regulation of gene expression, synthesis of RNA molecules and their modification. Proteins are important components of nuclear bodies and are involved in the maintenance of the nuclear architecture, transport across the nuclear envelope and cell division. Given their importance, the current poor knowledge of plant nuclear proteins and their dynamics during the cell's life and division is striking. Several factors hamper the analysis of the plant nuclear proteome, but the most critical seems to be the contamination of nuclei by cytosolic material during their isolation. With the availability of an efficient protocol for the purification of plant nuclei, based on flow cytometric sorting, contamination by cytoplasmic remnants can be minimized. Moreover, flow cytometry allows the separation of nuclei in different stages of the cell cycle (G1, S, and G2). This strategy has led to the identification of large number of nuclear proteins from barley (Hordeum vulgare), thus triggering the creation of a dedicated database called UNcleProt, http://barley.gambrinus.ueb.cas.cz/ .

Microscale affinity purification of trypsin reduces background peptides in matrix-assisted laser desorption/ionization mass spectrometry of protein digests.

The use of trypsin for protein digestion is hampered by its autolysis and low thermostability. Chemical modifications have been employed to stabilize the enzyme. Modified trypsin (e.g. methylated) usually enables performing digestions at elevated temperatures, but it still produces autolytic peptides. In this work, unmodified bovine trypsin was subjected to a microscale affinity chromatography on Arginine Sepharose (ASE) or Benzamidine Sepharose (BSE), which utilized the principle of active-site ligand binding. Trypsin was retained on the sorbents in ammonium bicarbonate as a binding buffer. After washings to remove unbound impurities, the enzyme was eluted by arginine as a free ligand (from ASE) or by diluted hydrochloric acid (from BSE). MALDI-TOF mass spectrometry confirmed removal of large molecular fragments as well as autolytic and other background peptides. Consequently, the purified trypsin was tested for its performance in procedures of in-gel digestion of protein standards and selected urinary proteins from real samples. It has been shown that the affinity purification of trypsin decreases significantly the number of unmatched peptides in peptide mass fingerprints. The presence of arginine in the digestion buffer was found to reduce intensity of autolytic peptides. As a result, the described purification procedure is applicable in a common proteomic routine.

Novel potent pharmacological cyclin-dependent kinase inhibitors.

Progression of the cell cycle is controlled by various activating and inhibiting cellular factors. The subtle balance between these counteracting regulators in normal cells ensures proper cell cycle progression and facilitates cellular responses to a variety of stress stimuli. Key activators include cyclin-dependent kinases (CDKs) and, consequently, loss or inactivation of CDK inhibitors contributes to the escape of cancer cells from cell cycle control and hyperactivation of CDKs occurs in various neurodegenerative disorders. However, these adverse effects may be compensated by pharmacological counterparts. Inhibitors of CDKs representing various classes of compounds with diverse CDK inhibitory patterns have been developed, but inhibitors that have high selectivity and offer highly targeted activity against both cell cycle and transcriptional CDKs are of particular interest. This review focuses on pharmacological CDK inhibitors that have entered clinical trials and some compounds that have been evaluated preclinically. Recent discoveries in cell cycle regulation have provided rationales for clinical applications of CDK inhibitors in both monotherapeutic and combined therapeutic regimens.