pH modulates interaction of 14-3-3 proteins with pollen plasma membrane H+ ATPases independently from phosphorylation.

Pollen grains transport the sperm cells through the style tissue via a fast-growing pollen tube to the ovaries where fertilization takes place. Pollen tube growth requires a precisely regulated network of cellular as well as molecular events including the activity of the plasma membrane H+ ATPase, which is known to be regulated by reversible protein phosphorylation and subsequent binding of 14-3-3 isoforms. Immunodetection of the phosphorylated penultimate threonine residue of the pollen plasma membrane H+ ATPase (LilHA1) of Lilium longiflorum pollen revealed a sudden increase in phosphorylation with the start of pollen tube growth. In addition to phosphorylation, pH modulated the binding of 14-3-3 isoforms to the regulatory domain of the H+ ATPase, whereas metabolic components had only small effects on 14-3-3 binding, as tested with in vitro assays using recombinant 14-3-3 isoforms and phosphomimicking substitutions of the threonine residue. Consequently, local H+ influxes and effluxes as well as pH gradients in the pollen tube tip are generated by localized regulation of the H+ ATPase activity rather than by heterogeneous localized distribution in the plasma membrane.

A novel FRET peptide assay reveals efficient Helicobacter pylori HtrA inhibition through zinc and copper binding.

Helicobacter pylori (H. pylori) secretes the chaperone and serine protease high temperature requirement A (HtrA) that cleaves gastric epithelial cell surface proteins to disrupt the epithelial integrity and barrier function. First inhibitory lead structures have demonstrated the essential role of HtrA in H. pylori physiology and pathogenesis. Comprehensive drug discovery techniques allowing high-throughput screening are now required to develop effective compounds. Here, we designed a novel fluorescence resonance energy transfer (FRET) peptide derived from a gel-based label-free proteomic approach (direct in-gel profiling of protease specificity) as a valuable substrate for H. pylori HtrA. Since serine proteases are often sensitive to metal ions, we investigated the influence of different divalent ions on the activity of HtrA. We identified Zn++ and Cu++ ions as inhibitors of H. pylori HtrA activity, as monitored by in vitro cleavage experiments using casein or E-cadherin as substrates and in the FRET peptide assay. Putative binding sites for Zn++ and Cu++ were then analyzed in thermal shift and microscale thermophoresis assays. The findings of this study will contribute to the development of novel metal ion-dependent protease inhibitors, which might help to fight bacterial infections.

In Vivo Cross-Linking to Analyze Transient Protein-Protein Interactions.

Cross-linking converts noncovalent interactions between proteins into covalent bonds. The now artificially fused molecules are stable during purification steps (e.g., immunoprecipitation). In combination with a variety of techniques, including Western blotting, mass spectrometry (MS), and bioinformatics, this technology provides improved opportunities for modelling structural details of functional complexes in living cells and protein-protein interaction networks. The presented strategy of immunoaffinity purification and mass spectrometry (AP-MS) coupled with in vivo cross-linking can easily be adapted as a robust workflow in interactome analyses of various species, also nonmodel organisms.

Sucrose-induced Receptor Kinase 1 is Modulated by an Interacting Kinase with Short Extracellular Domain.

Sucrose as a product of photosynthesis is the major carbohydrate translocated from photosynthetic leaves to growing nonphotosynthetic organs such as roots and seeds. These growing tissues, besides carbohydrate supply, require uptake of water through aquaporins to enhance cell expansion during growth. Previous work revealed Sucrose Induced Receptor Kinase, SIRK1, to control aquaporin activity via phosphorylation in response to external sucrose stimulation. Here, we present the regulatory role of AT3G02880 (QSK1), a receptor kinase with a short external domain, in modulation of SIRK1 activity. Our results suggest that SIRK1 autophosphorylates at Ser-744 after sucrose treatment. Autophosphorylated SIRK1 then interacts with and transphosphorylates QSK1 and QSK2. Upon interaction with QSK1, SIRK1 phosphorylates aquaporins at their regulatory C-terminal phosphorylation sites. Consequently, in root protoplast swelling assays, the qsk1qsk2 mutant showed reduced water influx rates under iso-osmotic sucrose stimulation, confirming an involvement in the same signaling pathway as the receptor kinase SIRK1. Large-scale phosphoproteomics comparing single mutant sirk1, qsk1, and double mutant sirk1 qsk1 revealed that aquaporins were regulated by phosphorylation depending on an activated receptor kinase complex of SIRK1, as well as QSK1. QSK1 thereby acts as a coreceptor stabilizing and enhancing SIRK1 activity and recruiting substrate proteins, such as aquaporins.

Dissecting the subcellular membrane proteome reveals enrichment of H+ (co-)transporters and vesicle trafficking proteins in acidic zones of Chara internodal cells.

The Characeae are multicellular green algae with very close relationship to land plants. Their internodal cells have been the subject of numerous (electro-)physiological studies. When exposed to light, internodal cells display alternating bands of low and high pH along their surface in order to facilitate carbon uptake required for photosynthesis. Here we investigated for the first time the subcellular membrane protein composition of acidic and alkaline regions in internodal cells of Chara australis R. Br. using MS-proteomics. The identified peptides were annotated to Chara unigenes using a custom-made Chara database generated from a transcriptome analysis and to orthologous Arabidopsis genes using TAIR (The Arabidopsis Information Resource) database. Apart from providing the first public-available, functionally-annotated sequence database for Chara australis, the proteome study, which is supported by immunodetection, identified several membrane proteins associated with acidic regions that contain a high density of specific plasma membrane (PM) invaginations, the charasomes, which locally increase the membrane area to overcome diffusion limitation in membrane transport. An increased abundance of PM H+ ATPases at charasomes is consistent with their role in the acidification of the environment, but the characean PM H+ ATPase sequence suggests a different regulation compared to higher plant PM H+ ATPases. A higher abundance of H+ co-transporters in the charasome-rich, acidic regions possibly reflects enhanced uptake of ions and nutrients. The increase in mitochondrial proteins confirms earlier findings about the accumulation of cortical mitochondria in the acidic zones. The significant enrichment of clathrin heavy chains and clathrin adaptor proteins as well as other proteins involved in trafficking indicate a higher activity of membrane transport in the charasome-rich than in charasome-poor areas. New and unexpected data, for instance the upregulation and abundance of vacuolar transporters correlating with the charasome-rich, acidic cell regions account for new perspectives in the formation of charasomes.

Identification of Cargo for Adaptor Protein (AP) Complexes 3 and 4 by Sucrose Gradient Profiling.

Intracellular vesicle trafficking is a fundamental process in eukaryotic cells. It enables cellular polarity and exchange of proteins between subcellular compartments such as the plasma membrane or the vacuole. Adaptor protein complexes participate in the vesicle formation by specific selection of the transported cargo. We investigated the role of the adaptor protein complex 3 (AP-3) and adaptor protein complex 4 (AP-4) in this selection process by screening for AP-3 and AP-4 dependent cargo proteins. Specific cargo proteins are expected to be mis-targeted in knock-out mutants of adaptor protein complex components. Thus, we screened for altered distribution profiles across a density gradient of membrane proteins in wild type versus ap-3ß and ap-4ß knock-out mutants. In ap-3ß mutants, especially proteins with transport functions, such as aquaporins and plasma membrane ATPase, as well as vesicle trafficking proteins showed differential protein distribution profiles across the density gradient. In the ap-4ß mutant aquaporins but also proteins from lipid metabolism were differentially distributed. These proteins also showed differential phosphorylation patterns in ap-3ß and ap-4ß compared with wild type. Other proteins, such as receptor kinases were depleted from the AP-3 mutant membrane system, possibly because of degradation after mis-targeting. In AP-4 mutants, membrane fractions were depleted for cytochrome P450 proteins, cell wall proteins and receptor kinases. Analysis of water transport capacity in wild type and mutant mesophyll cells confirmed aquaporins as cargo proteins of AP-3 and AP-4. The combination of organelle density gradients with proteome analysis turned out as a suitable experimental strategy for large-scale analyses of protein trafficking.

Lost in traffic? The K(+) channel of lily pollen, LilKT1, is detected at the endomembranes inside yeast cells, tobacco leaves, and lily pollen.

Fertilization in plants relies on fast growth of pollen tubes through the style tissue toward the ovules. This polarized growth depends on influx of ions and water to increase the tube's volume. K(+) inward rectifying channels were detected in many pollen species, with one identified in Arabidopsis. Here, an Arabidopsis AKT1-like channel (LilKT1) was identified from Lilium longiflorum pollen. Complementation of K(+) uptake deficient yeast mutants was only successful when the entire LilKT1 C-terminus was replaced by the AKT1 C-terminus. No signals were observed in the plasma membrane (PM) of pollen tubes after expression of fluorescence-tagged LilKT1 nor were any LilKT1-derived peptides detectable in the pollen PM by mass spectrometry analysis. In contrast, fluorescent LilKT1 partly co-localized with the lily PM H(+) ATPase LilHA2 in the PM of tobacco leaf cells, but exhibited a punctual fluorescence pattern and also sub-plasma membrane localization. Thus, incorporation of LilKT1 into the pollen PM seems tighter controlled than in other cells with still unknown trafficking signals in LilKT1's C-terminus, resulting in channel densities below detection limits. This highly controlled incorporation might have physiological reasons: an uncontrolled number of K(+) inward channels in the pollen PM will give an increased water influx due to the raising cytosolic K(+) concentration, and finally, causing the tube to burst.

De novo sequencing and analysis of the lily pollen transcriptome: an open access data source for an orphan plant species.

Pollen grains of Lilium longiflorum are a long-established model system for pollen germination and tube tip growth. Due to their size, protein content and almost synchronous germination in synthetic media, they provide a simple system for physiological measurements as well as sufficient material for biochemical studies like protein purifications, enzyme assays, organelle isolation or determination of metabolites during germination and pollen tube elongation. Despite recent progresses in molecular biology techniques, sequence information of expressed proteins or transcripts in lily pollen is still scarce. Using a next generation sequencing strategy (RNAseq), the lily pollen transcriptome was investigated resulting in more than 50 million high quality reads with a length of 90 base pairs. Sequenced transcripts were assembled and annotated, and finally visualized with MAPMAN software tools and compared with other RNAseq or genome data including Arabidopsis pollen, Lilium vegetative tissues and the Amborella trichopoda genome. All lily pollen sequence data are provided as open access files with suitable tools to search sequences of interest.

In vivo cross-linking combined with mass spectrometry analysis reveals receptor-like kinases and Ca(2+) signalling proteins as putative interaction partners of pollen plasma membrane H(+) ATPases.

During fertilisation in plants, pollen grains germinate and generate a pollen tube which grows through the style tissue to the egg apparatus delivering the two sperm cells for fertilisation. For this process, adaption to specific environmental conditions and communication between male and female organs are essential, requiring the sensing of internal and external signals which are translated into tube growth. The plasma membrane (PM) H(+) ATPase energises the pollen plasma membrane for nutrient, ion and water uptake, but additionally, its activity directly affects the germination frequency and drives the elongation of pollen tubes. A combination of in vivo cross-linking with para-formaldehyde, immunoaffinity purification of cross-linked PM H(+) ATPase complexes and subsequent mass spectrometry analysis revealed putative interaction partners of the PM H(+) ATPase of lily pollen, which are possibly involved in the perception and transduction of intra- and extracellular signals. Major interactions partners included (i) membrane-localised receptor-like kinases (RLKs) with the leucine-rich repeat RLKs (LRR-RLKs) forming the largest group, (ii) interacting protein kinases, phosphatases, WD-40 domain proteins and 14-3-3 proteins that may transduce intracellular, phosphorylation-dependent signals and (iii) specific cytosolic Ca(2+) signatures may be decoded by interacting Ca(2+) sensor proteins, calmodulin and calmodulin-like proteins, and Ca(2+)-dependent protein kinases, which were all identified as interaction partners of the PM H(+) ATPase in lily pollen. These identified interaction partners suggest new putative regulation mechanisms of the PM H(+) ATPase in general and new insights in regulating pollen tube growth rates in particular. Furthermore, the optimised experimental strategy can be applied to other non-model organisms to identify membrane protein interactions. BIOLOGICAL SIGNIFICANCE: Membrane proteomics is still very challenging due to the low abundance and poor solubility of membrane proteins. Furthermore, membrane protein interaction studies in a non-model organism like Lilium longiflorum require an unbiased preparation and detection approach. The presented strategy to identify putative interaction partners of the PM H(+) ATPase by using a combination of different biochemical techniques, i.e. in vivo crosslinking, immunoaffinity purification and mass spectrometry without the need of genetic engineering, transformation or other molecular biology techniques can be easily transferred to other protein interaction studies. The well characterised interaction of the PM H(+) ATPase with regulating 14-3-3 proteins served as an intrinsic control to proof the suitability and reliability of the presented strategy, whilst newly identified interaction partners may indicate novel regulation mechanisms of the PM H(+) ATPase.

Pollen cultivation and preparation for proteomic studies.

The quality of the collected experimental data very much depends on the quality of the biological starting material. Especially the proteome analysis of a highly dynamic system like the germinating and tube-growing pollen grain needs several precautions which allow an accurate and acceptable interpretation of the obtained results. Optimized protocols for pollen collection, storage, and in vitro culture as well as pollen organelle separations are described which help to obtain well-defined and reproducible experimental conditions for the subsequent proteomic analysis.

Pump up the volume - a central role for the plasma membrane H(+) pump in pollen germination and tube growth.

The plasma membrane H(+) ATPase is a member of the P-ATPase family transporting H(+) from the cytosol to the extracellular space and thus energizing the plasma membrane for the uptake of ions and nutrients. As a housekeeping gene, this protein can be detected in almost every plant cell including the exclusive expression of specific isoforms in pollen grains and tubes where its activity is a prerequisite for successful germination and growth of pollen tubes. This review summarizes the current knowledge on pollen PM H(+) ATPases and hypothesizes a central role for pollen-specific isoforms of this protein in tube growth. External as well as cytosolic signals from signal transduction and metabolic pathways are integrated by the PM H(+) ATPase and directly translated to tube growth rates, allocating the PM H(+) ATPase to an essential node in the signalling network of pollen tubes in their race to the ovule.

Sucrose-induced receptor kinase SIRK1 regulates a plasma membrane aquaporin in Arabidopsis.

The transmembrane receptor kinase family is the largest protein kinase family in Arabidopsis, and it contains the highest fraction of proteins with yet uncharacterized functions. Here, we present functions of SIRK1, a receptor kinase that was previously identified with rapid transient phosphorylation after sucrose resupply to sucrose-starved seedlings. SIRK1 was found to be an active kinase with increasing activity in the presence of an external sucrose supply. In sirk1 T-DNA insertional mutants, the sucrose-induced phosphorylation patterns of several membrane proteins were strongly reduced; in particular, pore-gating phosphorylation sites in aquaporins were affected. SIRK1-GFP fusions were found to directly interact with aquaporins in affinity pull-down experiments on microsomal membrane vesicles. Furthermore, protoplast swelling assays of sirk1 mutants and SIRK1-GFP expressing lines confirmed a direct functional interaction of receptor kinase SIRK1 and aquaporins as substrates for phosphorylation. A lack of SIRK1 expression resulted in the failure of mutant protoplasts to control water channel activity upon changes in external sucrose concentrations. We propose that SIRK1 is involved in the regulation of sucrose-specific osmotic responses through direct interaction with and activation of an aquaporin via phosphorylation and that the duration of this response is controlled by phosphorylation-dependent receptor internalization.

Dynamic adaption of metabolic pathways during germination and growth of lily pollen tubes after inhibition of the electron transport chain.

Investigation of the metabolome and the transcriptome of pollen of lily (Lilium longiflorum) gave a comprehensive overview of metabolic pathways active during pollen germination and tube growth. More than 100 different metabolites were determined simultaneously by gas chromatography coupled to mass spectrometry, and expressed genes of selected metabolic pathways were identified by next-generation sequencing of lily pollen transcripts. The time-dependent changes in metabolite abundances, as well as the changes after inhibition of the mitochondrial electron transport chain, revealed a fast and dynamic adaption of the metabolic pathways in the range of minutes. The metabolic state prior to pollen germination differed clearly from the metabolic state during pollen tube growth, as indicated by principal component analysis of all detected metabolites and by detailed observation of individual metabolites. For instance, the amount of sucrose increased during the first 60 minutes of pollen culture but decreased during tube growth, while glucose and fructose showed the opposite behavior. Glycolysis, tricarbonic acid cycle, glyoxylate cycle, starch, and fatty acid degradation were activated, providing energy during pollen germination and tube growth. Inhibition of the mitochondrial electron transport chain by antimycin A resulted in an immediate production of ethanol and a fast rearrangement of metabolic pathways, which correlated with changes in the amounts of the majority of identified metabolites, e.g. a rapid increase in γ-aminobutyric acid indicated the activation of a γ-aminobutyric acid shunt in the tricarbonic acid cycle, while ethanol fermentation compensated the reduced ATP production after inhibition of the oxidative phosphorylation.

Expression of the major mugwort pollen allergen Art v 1 in tobacco plants and cell cultures: problems and perspectives for allergen production in plants.

An economic and cheap production of large amounts of recombinant allergenic proteins might become a prerequisite for the common use of microarray-based diagnostic allergy assays which allow a component-specific diagnosis. A molecular pharming strategy was applied to express the major allergen of Artemisia vulgaris pollen, Art v 1, in tobacco plants and tobacco cell cultures. The original Art v 1 with its endogenous signal peptide which directs Art v 1 to the secretory pathway, was expressed in transiently transformed tobacco leaves but was lost in stable transformed tobacco plants during the alternation of generations. Using a light-regulated promoter and "hiding" the recombinant Art v 1 in the ER succeeded in expression of Art v 1 over three generations of tobacco plants and in cell cultures generated from stable transformed plants. However, the amounts of the recombinant allergen were sufficient for analysis but not high enough to allow an economic production. Although molecular pharming has been shown to work well for the production of non-plant therapeutic proteins, it might be less efficient for closely related plant proteins.

Pollen tubes and the physical world.

The primary goal of our previous opinion paper (Winship, L.J. et al. (2010) Trends Plant Sci. 15, 363-369) [1] was to put two models for the control of pollen tube growth on the same theoretical and biophysical footing, and to then test both for consistency with basic principles and with experimental data. Our central thesis, then and now, is that the biophysical and biochemical mechanisms that enable pollen tubes to grow and to respond to their environment evolved in a physical context constrained by known, inescapable principles. First, pressure is a scalar, not a vector quantity. Second, the water movement in and out of plant cells that generates pressure is passive, not active, and is controlled by differences in water potential. Here we respond to the issues raised by Zonia and Munnik (Trends Plant Sci. 2011; this issue) [2] in the light of new evidence concerning turgor pressure and pollen tube growth rates.

Glutamate receptor-like genes form Ca2+ channels in pollen tubes and are regulated by pistil D-serine.

Elevations in cytosolic free calcium concentration ([Ca(2+)](cyt)) constitute a fundamental signal transduction mechanism in eukaryotic cells, but the molecular identity of Ca(2+) channels initiating this signal in plants is still under debate. Here, we show by pharmacology and loss-of-function mutants that in tobacco and Arabidopsis, glutamate receptor-like channels (GLRs) facilitate Ca(2+) influx across the plasma membrane, modulate apical [Ca(2+)](cyt) gradient, and consequently affect pollen tube growth and morphogenesis. Additionally, wild-type pollen tubes grown in pistils of knock-out mutants for serine-racemase (SR1) displayed growth defects consistent with a decrease in GLR activity. Our findings reveal a novel plant signaling mechanism between male gametophyte and pistil tissue similar to amino acid-mediated communication commonly observed in animal nervous systems.

Identification of lily pollen 14-3-3 isoforms and their subcellular and time-dependent expression profile.

14-3-3 proteins are major regulators in plant development and physiology including primary metabolism and signal transduction pathways, typically via a phosphorylation-dependent interaction with a target protein. Four full-length 14-3-3 isoforms were identified in pollen grains of Lilium longiflorum by screening of a cDNA library and RACE (rapid amplification of cDNA ends)-PCR. Mass spectrometry analysis of partially purified 14-3-3s confirmed the presence of the four isoforms but also indicated the presence of additional, less abundant 14-3-3 isoforms in lily pollen. Separation of partially purified 14-3-3 proteins by two-dimensional gel electrophoresis resulted in nine spots that mainly contained the four major 14-3-3 isoforms. In a first step to examine putative physiological roles of specific 14-3-3 isoforms, their subcellular expression profile during pollen germination and tube growth was monitored using a characterized set of antibodies against 14-3-3 proteins with distinct crossreactivity. The abundance profile of 14-3-3 proteins associated with the cytosol, endomembranes (tonoplast, endoplasmic reticulum, Golgi, mitochondria) and plasma membrane showed high spatial-temporal dynamics. This indicates different targets of 14-3-3 proteins at different organelles and time points during pollen germination and growth.

Osmoregulation in Lilium pollen grains occurs via modulation of the plasma membrane H+ ATPase activity by 14-3-3 proteins.

To allow successful germination and growth of a pollen tube, mature and dehydrated pollen grains (PGs) take up water and have to adjust their turgor pressure according to the water potential of the surrounding stigma surface. The turgor pressure of PGs of lily (Lilium longiflorum) was measured with a modified pressure probe for simultaneous recordings of turgor pressure and membrane potential to investigate the relation between water and electrogenic ion transport in osmoregulation. Upon hyperosmolar shock, the turgor pressure decreased, and the plasma membrane (PM) hyperpolarizes in parallel, whereas depolarization of the PM was observed with hypoosmolar treatment. An acidification and alkalinization of the external medium was monitored after hyper- and hypoosmotic treatments, respectively, and pH changes were blocked by vanadate, indicating a putative role of the PM H(+) ATPase. Indeed, an increase in PM-associated 14-3-3 proteins and an increase in PM H(+) ATPase activity were detected in PGs challenged by hyperosmolar medium. We therefore suggest that in PGs the PM H(+) ATPase via modulation of its activity by 14-3-3 proteins is involved in the regulation of turgor pressure.

Under pressure, cell walls set the pace.

Significant controversy still swirls around the regulation of extension by tip-growing cells, particularly during stable, oscillatory growth of pollen tubes. One explanation proposes that turgor pressure is both the controlling and driving force. We refute this hypothesis on theoretical and evidentiary grounds. Direct measurement of intracellular pressure reveals constant turgor even as growth rates change. Measured ion fluxes, notably potassium, are insufficient to account for the requisite osmotic changes. Water movement, and hence pressure gradients, occur throughout the cell, unrestricted to local domains. Increases in hydrostatic pressure alone would force water out of the cell rather than cause increased growth. We have recently demonstrated concomitant changes in the apical cell wall that account fully for observed changes in growth rate.

The pollen organelle membrane proteome reveals highly spatial-temporal dynamics during germination and tube growth of lily pollen.

As a first step in understanding the membrane-related dynamics during pollen grain germination and subsequent tube growth, the changes in protein abundance of membrane and membrane-associated proteins of 5 different membrane/organelle fractions were studied at physiologically important stages (0, 10, 30, 60, and 240 min) of Lilium longiflorum pollen in vitro culture. Proteins of each fraction and time point were identified by 'shot-gun' proteomics (LC-MS/MS). Analysis of more than 270 identified proteins revealed an increase in the abundance of proteins involved in cytoskeleton, carbohydrate and energy metabolism, as well as ion transport before pollen grain germination (10-30 min), whereas proteins involved in membrane/protein trafficking, signal transduction, stress response and protein biosynthesis decreased in abundance during this time. Proteins of amino acids and lipids/steroids metabolism, proteolysis, transcription, cell wall biosynthesis as well as nutrient transport showed a time-independent abundance profile. These spatiotemporal patterns were confirmed by immunodetection of specific proteins of the cellular processes membrane/protein trafficking and ion transport. Our results reveal major protein rearrangements at endomembranes and the plasma membrane before and as the pollen grains start tube growth. The spatiotemporal protein abundance changes correlate with the underlying developmental and physiological processes of the germinating pollen grain.