Caspase-8-mediated cleavage inhibits IRF-3 protein by facilitating its proteasome-mediated degradation

Interferon regulatory factor 3 (IRF-3) plays a central role in inducing the expression of cellular antiviral genes, including the interferon-β gene, in response to Pattern Recognition Receptors. IRF-3 is targeted for proteasome-mediated degradation, which modulates the strength and duration of the innate immune responses that depend on it. We have found that caspase-8, which is activated by cytosolic RIG-I-dependent signaling, catalyzes an essential intermediate step in the ubiquitination and proteasome-mediated degradation of IRF-3. Mutation of a consensus cleavage site within IRF-3 generates a form that is not cleaved by caspase-8 and that is protected from ubiquitination and degradation. An in vitro assay confirms the direct action of caspase-8 cleavage on IRF-3. We also show that caspase-8-mediated cleavage of IRF-3 helps to modulate dsRNA-dependent gene induction.     

Cytokinin treatment of embryos inhibits the synthesis of chloroplast proteins in Norway spruce

A pulse treatment of Norway spruce (Picea abies (L.) Karst) embryos with the cytokinin N⁶-benzyladenine induces the formation of adventitious buds from subepidermal cells in the hypocotyl and cotyledons. In addition the treatment also inhibits elongation growth, a key process during germination. In this report we demonstrate that these effects on development of the plant are associated with a suppression of the accumulation of several major chloroplast proteins during germination. These proteins include the large subunit of ribulose bisphosphate/carboxylase oxygenase, two subunits of the chloroplast ATPase, protochlorophyllide reductase and a 23000-M_r component of photosystem II. For two nuclear-encoded proteins, the small subunit of ribulose bisphosphate carboxylase/oxygenase and the light-harvesting chlorophyll a/b-binding protein, a corresponding suppression of the increase in the steady-state amounts of mRNA is recorded. The suppression of chloroplast protein synthesis is consistant with the previously documented delay in greening that results from cytokinin treatment, but the effect is opposite to that found in other plants, where cytokinins promote the synthesis of chloroplast proteins, and stimulate chloroplast biogenesis. We believe that this difference is explained by the cytokinin primarily suppressing organ development, and a strict dependance of chloroplast biogenesis on the developmental state of the organs.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - CF1 coupling-factor 1 of chloroplast ATPase - LHCP light-harvesting chlorophyll a/b-binding protein - LSU large subunit of Rubisco - NADPH-protochlorophyllide oxidoreductase Pchlide reductase - SDS sodium dodecyl sulfate - SSU small subunit of Rubisco We thank K. Hutchison (Dept. of Biochemistry, University of Maine, Orono, Maine, USA) and P. Gustafsson (Dept. of Plant Physiology, University of Umeå, Sweden) for providing the Larix and Pinus clones, and M. Ryberg (Dept. of Plant Physiology, University of Göteborg, Sweden), R. Ölmüller (Botanisches Institut, Universität München, FRG) and W. Lockau (Institut für Botanik, Universität Regensburg, FRG), for the gift of antisera towards Pchlide reductase, RuBPCase and LHCP, and ATPase, respectively. Supported by the Swedish Council for Forestry and Agricultural Research and the Swedish Natural Sciences Research Council.     

Cytokinin promotes growth cessation in the Arabidopsis root

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Cytokinin levels in leaves, leaf exudate and shoot apical meristem of Arabidopsis thaliana during floral transition

Understanding the complete picture of floral transition is still impaired by the fact that physiological studies mainly concern plant species whose genetics is poorly known, and vice versa. Arabidopsis thaliana has been successfully used to unravel signalling pathways by genetic and molecular approaches, but analyses are still required to determine the physiological signals involved in the control of floral transition. In this work, the putative role of cytokinins was investigated using vegetative plants of Arabidopsis (Columbia) induced to flower synchronously by a single 22 h long day. Cytokinins were analysed in leaf extracts, leaf phloem exudate and in the shoot apical meristem at different times during floral transition. It was found that, in both the leaf tissues and leaf exudate, isopentenyladenine forms of cytokinins increased from 16 h after the start of the long day. At 30 h, the shoot apical meristem of induced plants contained more isopentenyladenine and zeatin than vegetative controls. These cytokinin increases correlate well with the early events of floral transition.     

Global views of proteasome-mediated degradation by mass spectrometry

Introduction: Degradation of proteins by cellular proteasomes is critical for the fidelity of protein homeostasis and proper cell function. Indeed, perturbations in proteasome function, as well as the degradation of specific substrates, are associated with a variety of human diseases. Yet, monitoring and analyzing protein degradation in a high throughput manner in physiology and pathology remains limited.

Areas covered: Here we discuss several of the recently developed mass spectrometry-based methods for studying proteasome-mediated cellular degradation and discuss their advantages and limitations. We highlight Mass Spectrometry Analysis of Proteolytic Peptides (MAPP), a method designed to purify and identify proteasome-cleaved cellular proteins as a novel approach in molecular and clinical profiling of human disease.

Expert opinion: The recent improvement of proteomics technologies now offers an unprecedented ability to study disease in clinical settings. Expanding clinical studies to include the degradation landscape will provide a new resolution to complement the cellular proteome. In turn, this holds promise to provide both new disease targets and novel peptide biomarkers which will further enhance personalized proteomics.     

Proteomic approaches to identifying carbonylated proteins in brain tissue

Oxidative stress plays a critical role in the pathogenesis of a number of diseases. The carbonyl end products of protein oxidation are among the most commonly measured markers of oxidation in biological samples. Protein carbonyl functional groups may be derivatized with 2,4-dinitrophenylhydrazine (DNPH) to render a stable 2,4-dinitrophenylhydrazone-protein (DNP-protein) and the carbonyl contents of individual proteins then determined by two-dimensional electrophoresis followed by immunoblotting using specific anti-DNP antibodies. Unfortunately, derivatization of proteins with DNPH could affect their mass spectrometry (MS) identification. This problem can be overcome using nontreated samples for protein identification. Nevertheless, derivatization could also affect their mobility, which might be solved by performing the derivatization step after the initial electrophoresis. Here, we compare two-dimensional redox proteome maps of mouse cerebellum acquired by performing the DNPH derivatization step before or after electrophoresis and detect differences in protein patterns. When the same approach is used for protein detection and identification, both methods were found to be useful to identify carbonylated proteins. However, whereas pre-DNPH derivatized proteins were successfully analyzed, high background staining complicated the analysis when the DNPH reaction was performed after transblotting. Comparative data on protein identification using both methods are provided.     

ABA inhibits entry into stomatal‐lineage development in Arabidopsis leaves

The number and density of stomata are controlled by endogenous and environmental factors. Despite recent advances in our understanding of stomatal development, mechanisms which prevent stomatal‐lineage entry remain unclear. Here, we propose that abscisic acid (ABA), a phytohormone known to induce stomatal closure, limits initiation of stomatal development and induces enlargement of pavement cells in Arabidopsis cotyledons. An ABA‐deficient aba2‐2 mutant had an increased number/proportion of stomata within a smaller cotyledon, as well as reduced expansion of pavement cells. This tendency was reversed after ABA application or in an ABA over‐accumulating cyp707a1cyp707a3 doublemutant. Our time course analysis revealed that aba2‐2 shows prolonged formation of meristemoids and guard mother cells, both precursors of stoma. This finding is in accordance with prolonged gene expression of SPCH and MUTE, master regulators for stomatal formation, indicating that ABA acts upstream of these genes. Only aba2‐2 mute, but not aba2‐2 spch double mutant showed additive phenotypes and displayed inhibition of pavement cell enlargement with increased meristemoid number, indicating that ABA action on pavement cell expansion requires the presence of stomatal‐lineage cells.     

'Senescence-associated vacuoles' are involved in the degradation of chloroplast proteins in tobacco leaves

Massive degradation of photosynthetic proteins is the hallmark of leaf senescence; however the mechanism involved in chloroplast protein breakdown is not completely understood. As small 'senescence-associated vacuoles' (SAVs) with intense proteolytic activity accumulate in senescing leaves of soybean and Arabidopsis, the main goal of this work was to determine whether SAVs are involved in the degradation of chloroplastic components. SAVs with protease activity were readily detected through confocal microscopy of naturally senescing leaves of tobacco (Nicotiana tabacum L.). In detached leaves incubated in darkness, acceleration of the chloroplast degradation rate by ethylene treatment correlated with a twofold increase in the number of SAVs per cell, compared to untreated leaves. In a tobacco line expressing GFP targeted to plastids, GFP was re-located to SAVs in senescing leaves. SAVs were isolated by sucrose density gradient centrifugation. Isolated SAVs contained chloroplast-targeted GFP and the chloroplast stromal proteins Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) and glutamine synthetase, but lacked the thylakoid proteins D1 and light-harvesting complex II of the photosystem II reaction center and photosystem II antenna, respectively. In SAVs incubated at 30 degrees C, there was a steady decrease in Rubisco levels, which was completely abolished by addition of protease inhibitors. These results indicate that SAVs are involved in degradation of the soluble photosynthetic proteins of the chloroplast stroma during senescence of leaves.     

Arabidopsis thaliana LSM proteins function in mRNA splicing and degradation

Sm-like (Lsm) proteins have been identified in all organisms and are related to RNA metabolism. Here, we report that Arabidopsis nuclear AtLSM8 protein, as well as AtLSM5, which localizes to both the cytoplasm and nucleus, function in pre-mRNA splicing, while AtLSM5 and the exclusively cytoplasmic AtLSM1 contribute to 5′–3′ mRNA decay. In lsm8 and sad1/lsm5 mutants, U6 small nuclear RNA (snRNA) was reduced and unspliced mRNA precursors accumulated, whereas mRNA stability was mainly affected in plants lacking AtLSM1 and AtLSM5. Some of the mRNAs affected in lsm1a lsm1b and sad1/lsm5 plants were also substrates of the cytoplasmic 5′–3′ exonuclease AtXRN4 and of the decapping enzyme AtDCP2. Surprisingly, a subset of substrates was also stabilized in the mutant lacking AtLSM8, which supports the notion that plant mRNAs are actively degraded in the nucleus. Localization of LSM components, purification of LSM-interacting proteins as well as functional analyses strongly suggest that at least two LSM complexes with conserved activities in RNA metabolism, AtLSM1-7 and AtLSM2-8, exist also in plants.     

Cytokinin signaling regulates two-stage inflorescence arrest in Arabidopsis

To maximize reproductive success, flowering plants must correctly time entry and exit from the reproductive phase. While much is known about mechanisms that regulate initiation of flowering, end-of-flowering remains largely uncharacterized. End-of-flowering in Arabidopsis (Arabidopsis thaliana) consists of quasi-synchronous arrest of inflorescences, but it is unclear how arrest is correctly timed with respect to environmental stimuli and reproductive success. Here, we showed that Arabidopsis inflorescence arrest is a complex developmental phenomenon, which includes the arrest of the inflorescence meristem (IM), coupled with a separable “floral arrest” of all unopened floral primordia; these events occur well before visible inflorescence arrest. We showed that global inflorescence removal delays both IM and floral arrest, but that local fruit removal only delays floral arrest, emphasizing their separability. We tested whether cytokinin regulates inflorescence arrest, and found that cytokinin signaling dynamics mirror IM activity, while cytokinin treatment can delay both IM and floral arrest. We further showed that gain-of-function cytokinin receptor mutants can delay IM and floral arrest; conversely, loss-of-function mutants prevented the extension of flowering in response to inflorescence removal. Collectively, our data suggest that the dilution of cytokinin among an increasing number of sink organs leads to end-of-flowering in Arabidopsis by triggering IM and floral arrest.

The phytohormone cytokinin regulates multiple distinct developmental events at the end of flowering in Arabidopsis thaliana.     

Cytokinin synthesis is higher in the Arabidopsis amp1 mutant

Cytokinins are involved in plant cell proliferation leading to plant growth and morphogenesis. Earlier we described a mutant of Arabidopsis thaliana, amp1, that had five times higher levels of cytokinin and had a number of pleiotropic phenotypes, including increased cell proliferation and de-etiolated growth in the dark. While these phenotypes were correlated with higher levels of cytokinin, the actual mechanism of how cytokinin is elevated was not elucidated before. In order to understand if the increased cytokinin is a result of increased biosynthesis or decreased degradation we have compared the synthesis of cytokinins from radiolabelled adenine and the degradation of zeatin ribosides and other cytokinins between amp1 and wild type plants. The degradation of the hormone is not affected in the mutant but there is a 4 to 6 fold increase in cytokinin synthesis compared to the wild type. Because the amp1 mutant is recessive we hypothesise that the AMP1 product negatively regulates cytokinin production.     

LOV domain-containing F-box proteins: light-dependent protein degradation modules in Arabidopsis

Plants constantly survey the surrounding environment using several sets of photoreceptors. They can sense changes in the quantity (=intensity) and quality (=wavelength) of light and use this information to adjust their physiological responses, growth, and developmental patterns. In addition to the classical photoreceptors, such as phytochromes, cryptochromes, and phototropins, ZEITLUPE (ZTL), FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1), and LOV KELCH PROTEIN 2 (LKP2) proteins have been recently identified as blue-light photoreceptors that are important for regulation of the circadian clock and photoperiodic flowering. The ZTL/FKF1/LKP2 protein family possesses a unique combination of domains: a blue-light-absorbing LOV (Light, Oxygen, or Voltage) domain along with domains involved in protein degradation. Here, we summarize recent advances in our understanding of the function of the Arabidopsis ZTL/FKF1/LKP2 proteins. We summarize the distinct photochemical properties of their LOV domains and discuss the molecular mechanisms by which the ZTL/FKF1/LKP2 proteins regulate the circadian clock and photoperiodic flowering by controlling blue-light-dependent protein degradation.     

Cell-Type-Specific Cytokinin Distribution within the Arabidopsis Primary Root Apex

Cytokinins (CKs) play a crucial role in many physiological and developmental processes at the levels of individual plant components (cells, tissues, and organs) and by coordinating activities across these parts. High-resolution measurements of intracellular CKs in different plant tissues can therefore provide insights into their metabolism and mode of action. Here, we applied fluorescence-activated cell sorting of green fluorescent protein (GFP)-marked cell types, combined with solid-phase microextraction and an ultra-high-sensitivity mass spectrometry (MS) method for analysis of CK biosynthesis and homeostasis at cellular resolution. This method was validated by series of control experiments, establishing that protoplast isolation and cell sorting procedures did not greatly alter endogenous CK levels. The MS-based method facilitated the quantification of all the well known CK isoprenoid metabolites in four different transgenic Arabidopsis thaliana lines expressing GFP in specific cell populations within the primary root apex. Our results revealed the presence of a CK gradient within the Arabidopsis root tip, with a concentration maximum in the lateral root cap, columella, columella initials, and quiescent center cells. This distribution, when compared with previously published auxin gradients, implies that the well known antagonistic interactions between the two hormone groups are cell type specific.