Optical Slices Research Articles

Proteomic Pathway Analysis Reveals Inflammation Increases Myeloid-Derived Suppressor Cell Resistance to Apoptosis

Myeloid-derived suppressor cells (MDSC) accumulate in patients and animals with cancer where they mediate systemic immune suppression and obstruct immune-based cancer therapies. We have previously demonstrated that inflammation, which frequently accompanies tumor onset and progression, increases the rate of accumulation and the suppressive potency of MDSC. To determine how inflammation enhances MDSC levels and activity we used mass spectrometry to identify proteins produced by MDSC induced in highly inflammatory settings. Proteomic pathway analysis identified the Fas pathway and caspase network proteins, leading us to hypothesize that inflammation enhances MDSC accumulation by increasing MDSC resistance to Fas-mediated apoptosis. The MS findings were validated and extended by biological studies. Using activated caspase 3 and caspase 8 as indicators of apoptosis, flow cytometry, confocal microscopy, and Western blot analyses demonstrated that inflammation-induced MDSC treated with a Fas agonist contain lower levels of activated caspases, suggesting that inflammation enhances resistance to Fas-mediated apoptosis. Resistance to Fas-mediated apoptosis was confirmed by viability studies of MDSC treated with a Fas agonist. These results suggest that an inflammatory environment, which is frequently present in tumor-bearing individuals, protects MDSC against extrinsic-induced apoptosis resulting in MDSC with a longer in vivo half-life, and may explain why MDSC accumulate more rapidly and to higher levels in inflammatory settings.

Activity of BKCa Channel Is Modulated by Membrane Cholesterol Content and Association with Na+/K+-ATPase in Human Melanoma IGR39 Cells

Interaction of large conductance Ca(2+)- and voltage-activated K(+) (BK(Ca)) channels with Na(+)/K(+)-ATPase, caveolin-1, and cholesterol was studied in human melanoma IGR39 cells. Functional BK(Ca) channels were enriched in caveolin-rich and detergent-resistant membranes, i.e. rafts, and blocking of the channels by a specific BK(Ca) blocker paxilline reduced proliferation of the cells. Disruption of rafts by selective depletion of cholesterol released BK(Ca) channels from these domains with a consequent increase in their activity. Consistently, cholesterol enrichment of the cells increased the proportion of BK(Ca) channels in rafts and decreased their activity. Immunocytochemical analysis showed that BK(Ca) channels co-localize with Na(+)/K(+)-ATPase in a cholesterol-dependent manner, thus suggesting their co-presence in rafts. Supporting this, ouabain, a specific blocker of Na(+)/K(+)-ATPase, inhibited BK(Ca) whole-cell current markedly in control cells but not in cholesterol-depleted ones. This inhibition required the presence of external Na(+). Collectively, these data indicate that the presence of Na(+)/K(+)-ATPase in rafts is essential for efficient functioning of BK(Ca) channels, presumably because the pump maintains a low intracellular Na(+) proximal to the BK(Ca) channel. In conclusion, cholesterol could play an important role in cellular ion homeostasis and thus modulate many cellular functions and cell proliferation.

73 LIPID CONTENT AND CRYOTOLERANCE OF PORCINE EMBRYOS CULTURED WITH PHENAZINE ETHOSULFATE

In this study, the addition of phenazine ethosulfate (PES) to culture medium was investigated for its effect on cytoplasmic lipid content in cultured pig embryo and survival after open pulled straw (OPS) vitrification (Vajta et al. 1997 Acta Vet. Scand. 38, 363–366). In addition, in cultured blastocysts, the total cell number per blastocyst and the degree of apoptosis were assessed. Porcine zygotes were cultured up to the blastocyst stage in NCSU-23 medium supplemented with 0 (control, n = 146) or 0.05 μM PES (n = 150). To evaluate the lipid content in embryos, we employed Nile Red (NR), a fluorescent dye specific for intracellular lipids (Genicot et al. 2005 Theriogenology 63, 1181–1194). We measured the amount of fluorescence originating from NR using LSM 510 Meta Zeiss confocal microscope and ImageJ version 1.38x software (National Institutes of Health, Bethesda, MD, USA) and an Integrated Density (ID) parameter. The total amount of fluorescence per embryo (TF), proportional to the amount of lipids, was calculated as the sum of ID measured for all optical slices in each individual z-stack. Blastocysts that were cultured with (n = 48) or without PES (n = 34) were vitrified using OPS technology. Results were analysed using chi-square, Fisher, and Student’s t-tests. The total number of cells and the percentage of TUNEL-positive nuclei of PES-treated blastocysts were significantly different than for the control group (43.6 v. 37.6; P < 0.05 and 1.6 v. 2.9; P < 0.01, respectively). Blastocysts stained with Nile Red fluorescent dye showed intracellular lipid mainly localised to the lipid droplets. They were present both in the embryoblast and trophoblast cells. Mean values of TF estimated for the experimental group was lower by ∼23% than those of the control group. Thereby, blastocysts of the control group possess a higher content of lipids then those found in the experimental group cultured in medium with 0.05 μM PES (P < 0.001). The survival rate of vitrified blastocysts was slightly enhanced, although not significantly, in the presence of PES compared to the PES-free group (44.8 and 37.1%, respectively). These results showed that culturing porcine embryos in medium with phenazine ethosulfate supplementation increased the total cell number per blastocyst and reduced the index of DNA fragmentation of cultured blastocysts. Use of PES in porcine culture medium reduced the cytoplasmic lipid content, as measured by fluorescence of blastocysts stained with Nile Red. However, the use of PES during in vitro culture had a limited effect on porcine blastocyst survival after vitrification. This study was partially supported by Grant NR 12 0036 06 from NCBiR, Poland.

A demonstration of the effectiveness of a single aberration correction per optical slice in beam scanned optically sectioning microscopes

In this paper we report the use of adaptive optics to correct for sample induced aberrations in optical microscopy, crucially comparing individual pixel-by-pixel correction against a single correction for an entire optical section. Sample induced optical aberrations in slices of rat brain tissue were corrected with a deformable membrane mirror. Using axial resolution measurements, we demonstrate that a single aberration correction per optical slice achieves around 80% of the maximum possible improvement compared to individual pixel-by-pixel correction in both confocal and multiphoton microscopy. A single aberration correction per depth, compared to pixel-by-pixel aberration correction, significantly decreases scan times and therefore reduces photobleaching and phototoxic effects enabling more rapid microscopy with active aberration correction. The results confirm that the use of a “look-up” table, based upon sample type and depth, may be the most practical way of implementing adaptive optic aberration correction in beam scanning optical sectioning microscopy.

Pannexin 2 Is Expressed by Postnatal Hippocampal Neural Progenitors and Modulates Neuronal Commitment

The pannexins (Panx1, -2, and -3) are a mammalian family of putative single membrane channels discovered through homology to invertebrate gap junction-forming proteins, the innexins. Because connexin gap junction proteins are known regulators of neural stem and progenitor cell proliferation, migration, and specification, we asked whether pannexins, specifically Panx2, play a similar role in the postnatal hippocampus. We show that Panx2 protein is differentially expressed by multipotential progenitor cells and mature neurons. Both in vivo and in vitro, Type I and IIa stem-like neural progenitor cells express an S-palmitoylated Panx2 species localizing to Golgi and endoplasmic reticulum membranes. Protein expression is down-regulated during neurogenesis in neuronally committed Type IIb and III progenitor cells and immature neurons. Panx2 is re-expressed by neurons following maturation. Protein expressed by mature neurons is not palmitoylated and localizes to the plasma membrane. To assess the impact of Panx2 on neuronal differentiation, we used short hairpin RNA to suppress Panx2 expression in Neuro2a cells. Knockdown significantly accelerated the rate of neuronal differentiation. Neuritic extension and the expression of antigenic markers of mature neurons occurred earlier in stable lines expressing Panx2 short hairpin RNA than in controls. Together, these findings describe an endogenous post-translational regulation of Panx2, specific to early neural progenitor cells, and demonstrate that this expression plays a role in modulating the timing of their commitment to a neuronal lineage.

3-D modelling of chloroplast structure under (Mg 2+) magnesium ion treatment. Relationship between thylakoid membrane arrangement and stacking

We performed for the first time three-dimensional (3D) modelling of the entire chloroplast structure. Stacks of optical slices obtained by confocal laser scanning microscope (CLSM) provided a basis for construction of 3D images of individual chloroplasts. We selected pea ( Pisum sativum) and bean ( Phaseolus vulgaris) chloroplasts since we found that they differ in thylakoid organization. Pea chloroplasts contain large distinctly separated appressed domains while less distinguished appressed regions are present in bean chloroplasts. Different magnesium ion treatments were used to study thylakoid membrane stacking and arrangement. In pea chloroplasts, as demonstrated by 3D modelling, the increase of magnesium ion concentration changed the degree of membrane appression from wrinkled continuous surface to many distinguished stacked areas and significant increase of the inter-grana area. On the other hand 3D models of bean chloroplasts exhibited similar but less pronounced tendencies towards formation of appressed regions. Additionally, we studied arrangements of thylakoid membranes and chlorophyll–protein complexes by various spectroscopic methods, Fourier-transform infrared spectroscopy (FTIR) among others. Based on microscopic and spectroscopic data we suggested that the range of chloroplast structure alterations under magnesium ions treatment is a consequence of the arrangement of supercomplexes. Moreover, we showed that stacking processes always affect the structural changes of chloroplast as a whole.

Confocal Laser Endomicroscopy: Technical Advances and Clinical Applications

Since its introduction in 2004, confocal laser endomicroscopy (CLE) has emerged as a valuable tool for gastrointestinal endoscopic imaging. Endomicroscopy enables the endoscopist to obtain real time in vivo histology during ongoing endoscopy thereby creating “optical biopsies.” To date, numerous studies have shown potential applications of endomicroscopy in the clinical setting, including in vivo diagnosis of esophageal squamous cell carcinoma, Barrett’s esophagus, celiac disease, and colonic polyps. Moreover, recent data suggest the potential application of endomicroscopy in the field of molecular imaging. Additionally, in recent months new applications and developments in the field of confocal imaging were introduced including endomicroscopy of the liver, pancreatic, and bile ducts. Furthermore, by introducing a new needle-based confocal imaging system, which is small enough to be introduced through a 22gauge puncture needle, a wide field for new applications of endomicroscopic imaging has been opened. Currently, 2 CE- and US Food and Drug Administration (FDA)approved devices for endomicroscopy are available (Figures 1 and 2; Supplementary Table 1). In this review, we introduce both systems and discuss new technical advances and clinical applications of CLE.

Embryonic Expression of Cyclooxygenase-2 Causes Malformations in Axial Skeleton

Cyclooxygenases (COXs) have important functions in various physiological and pathological processes. COX-2 expression is highly induced by a variety of stimuli and is observed during certain periods of embryonic development. In this report, the direct effect of COX-2 expression on embryonic development is examined in a novel COX-2 transgenic mouse model that ubiquitously expresses human COX-2 from the early stages of embryonic development. COX-2 transgenic fetuses exhibit severe skeletal malformations and die shortly after birth. Skeletal malformations are localized along the entire vertebral column and rib cage and are linked to defective formation of cartilage anlagen. The cartilage anlagen of axial skeleton fail to properly develop in transgenic embryos because of impaired precartilaginous sclerotomal condensation, which results from the reduction of cell number in the sclerotome. Despite the ubiquitous expression of COX-2, the number of apoptotic cells is highly increased in the sclerotome of transgenic embryos but not in other tissues, suggesting that it is a tissue-specific response. Therefore, the loss of sclerotomal cells due to an increased apoptosis is probably responsible for axial skeletal malformations in transgenic fetuses. In addition, the sclerotomal accumulation of p53 protein is observed in transgenic embryos, suggesting that COX-2 may induce apoptosis via the up-regulation of p53. Our results demonstrate that the aberrant COX-2 signaling during embryonic development is teratogenic and suggest a possible association of COX-2 with fetal malformations of unknown etiology.

Three-Dimensional Optical Sectioning by Scanning Confocal Electron Microscopy with a Stage-Scanning System

We evaluated the depth resolution of annular dark-field (ADF) scanning confocal electron microscopy (SCEM) with a stage-scanning system by observation of nanoparticles. ADF-SCEM is a three-dimensional (3D) imaging technique that we recently proposed. An ADF-SCEM instrument involves a pinhole aperture before a detector for rejecting electrons from the out-of-focal plane in a specimen and an annular aperture under the specimen for collecting only scattered electrons. The stage-scanning system enables us to directly obtain optical slice images perpendicular and parallel to an optical axis at a desired position. In particular, the parallel slices visualize the elongation of nanoparticles along the optical axis, which depends on the depth resolution. ADF-SCEM effectively reduced the elongation length of the nanoparticles sufficiently to demonstrate depth sectioning, in comparison with scanning transmission electron microscopy and bright-field SCEM. The experimentally obtained length was nearly equal to the theoretically estimated one from the probe size considering the experimental conditions. Furthermore, we applied this ADF-SCEM technique to analysis of the 3D position of catalytic nanoparticles on carbon nanostructures.

In Vivo Molecular Imaging of Somatostatin Receptors in Pancreatic Islet Cells and Neuroendocrine Tumors by Miniaturized Confocal Laser-Scanning Fluorescence Microscopy

The aim of the study was to evaluate real time in vivo molecular imaging of somatostatin receptors (sstrs) using a handheld miniaturized confocal laser scan microscope (CLM) in conjunction with fluorescein-labeled octreotate (OcF) in healthy mice and murine models of neuroendocrine tumors. For CLM a small rigid probe (diameter 7 mm) with an integrated single line laser (488 nm) was used (optical slice thickness 7 mum; lateral resolution 0.7 mum). OcF was synthesized via Fmoc solid-phase peptide synthesis and purified by HPLC showing high-affinity binding to the sstr2 (IC(50) 6.2 nmol). For in vitro evaluation, rat and human pancreatic cancer cells were used and characterized with respect to its sstr subtype expression and functional properties. For in vivo confocal imaging, healthy mouse pancreatic islet and renal tubular cells as well as immunoincompetent nude mice harboring sstr-expressing tumors were evaluated. Incubation of sstr-positive cells with OcF showed a specific time- and dose-dependent staining of sstr-positive cells. CLM showed rapid internalization and homogenous cytoplasmatic distribution. After systemic application to mice (n = 8), specific time-dependent internalization and cytoplasmatic distribution into pancreatic islet cells and tubular cells of the renal cortex was recorded. After injection in tumor-harboring nude mice (n = 8), sstr-positive cells selectively displayed a cell surface and cytoplasmatic staining. CLM-targeted biopsies detected sstr-positive tumor cells with a sensitivity of 87.5% and a specificity of 100% as correlated with ex vivo immunohistochemistry. CLM with OcF permits real-time molecular, functional, and morphological imaging of sstr-expressing cell structures, allowing the specific visualization of pancreatic islet cells and neuroendocrine tumors in vivo.

Whole-field measurement of three-dimensional stress by scattered-light photoelasticity with unpolarized light

In digital scattered-light photoelasticity with unpolarized light (DSLPUL), secondary principal stress direction ψ j and total relative phase retardation ρ jtot in a three-dimensional stressed model with rotation of the principal stress axes are obtained by measuring Stokes parameters of scattered light from optical slices. The present paper describes intelligibly the principle of DSLPUL, and then demonstrates that the ψ j and ρ jtot in a frozen stress sphere model are nondestructively measured over the entire field.

Peroxidasin Is Secreted and Incorporated into the Extracellular Matrix of Myofibroblasts and Fibrotic Kidney

Mammalian peroxidases are heme-containing enzymes that serve diverse biological roles, such as host defense and hormone biosynthesis. A mammalian homolog of Drosophila peroxidasin belongs to the peroxidase family; however, its function is currently unknown. In this study, we show that peroxidasin is present in the endoplasmic reticulum of human primary pulmonary and dermal fibroblasts, and the expression of this protein is increased during transforming growth factor-beta1-induced myofibroblast differentiation. Myofibroblasts secrete peroxidasin into the extracellular space where it becomes organized into a fibril-like network and colocalizes with fibronectin, thus helping to form the extracellular matrix. We also demonstrate that peroxidasin expression is increased in a murine model of kidney fibrosis and that peroxidasin localizes to the peritubular space in fibrotic kidneys. In addition, we show that this novel pathway of extracellular matrix formation is unlikely mediated by the peroxidase activity of the protein. Our data indicate that peroxidasin secretion represents a previously unknown pathway in extracellular matrix formation with a potentially important role in the physiological and pathological fibrogenic response.

Substantial energy expenditure for locomotion in ciliates verified by means of simultaneous measurement of oxygen consumption rate and swimming speed

In order to characterize the energy expenditure of Paramecium, we simultaneously measured the oxygen consumption rate, using an optic fluorescence oxygen sensor, and the swimming speed, which was evaluated by the optical slice method. The standard metabolic rate (SMR, the rate of energy consumption exclusively for physiological activities other than locomotion) was estimated to be 1.18x10(-6) J h(-1) cell(-1) by extrapolating the oxygen consumption rate into one at zero swimming speed. It was about 30% of the total energy consumed by the cell swimming at a mean speed of 1 mm s(-1), indicating that a large amount of the metabolic energy (about 70% of the total) is consumed for propulsive activity only. The mechanical power liberated to the environment by swimming Paramecium was calculated on the basis of Stokes' law. This power, termed Stokes power, was 2.2x10(-9) J h(-1) cell(-1), indicating extremely low efficiency (0.078%) in the conversion of metabolic power to propulsion. Analysis of the cost of transport (COT, the energy expenditure for translocation per units of mass and distance) revealed that the efficiency of energy expenditure in swimming increases with speed rather than having an optimum value within a wide range of forced swimming, as is generally found in fish swimming. These characteristics of energy expenditure would be unique to microorganisms, including Paramecium, living in a viscous environment where large dissipation of the kinetic energy is inevitable due to the interaction with the surrounding water.

RETRACTED: Expression and regulated nuclear transport of transducers of regulated CREB 1 in retinal ganglion cells

Calcium- and cAMP-dependent activation of CREB and transcription of cAMP-responsive element (CRE)-target genes play critical roles in various physiological and pathological conditions. TORCs (transducers of regulated CREB) represent a new family of conserved CREB coactivators that function as intracellular calcium- and cAMP-sensitive coincidence detectors, controlling the kinetics of CRE-mediated responses and long-term potentiation of synaptic transmission. Here we examined the expression and activity-dependent translocation of TORCs in adult retinal ganglion cells (RGCs), the primary target of acute retinal ischemic injury as well as chronic retinal degenerative diseases. We found that both mRNAs of TORC1 and TORC2, but not TORC3, were enriched in adult rat retina. Comparing with TORC2, TORC1 protein was highly and selectively expressed in RGCs. At resting condition, TORC1 protein was localized in the cytoplasm but not nucleus of RGCs. Activation of N-methyl-D-aspartate (NMDA) receptors by intravitreous injection of NMDA or increase of cAMP signaling by administration of forskolin triggered nuclear accumulation of TORC1. Furthermore, transient retinal ischemic injury resulted in peri-nuclear and nuclear accumulation of TORC1 as well as transcription of BDNF in RGCs. Our results demonstrate that TORC1 is enriched in RGCs and its subcellular location could be regulated by Ca(2+) and cAMP, suggesting that manipulation of TORC1 activity may promote survival of RGCs in some optic disease conditions.

High-Speed High-Resolution Imaging of Intercellular Immune Synapses Using Optical Tweezers

Imaging in any plane other than horizontal in a microscope typically requires a reconstruction from multiple optical slices that significantly decreases the spatial and temporal resolution that can be achieved. This can limit the precision with which molecular events can be detected, for example, at intercellular contacts. This has been a major issue for the imaging of immune synapses between live cells, which has generally required the reconstruction of en face intercellular synapses, yielding spatial resolution significantly above the diffraction limit and updating at only a few frames per minute. Strategies to address this issue have usually involved using artificial activating substrates such as antibody-coated slides or supported planar lipid bilayers, but synapses with these surrogate stimuli may not wholly resemble immune synapses between two cells. Here, we combine optical tweezers and confocal microscopy to realize generally applicable, high-speed, high-resolution imaging of almost any arbitrary plane of interest. Applied to imaging immune synapses in live-cell conjugates, this has enabled the characterization of complex behavior of highly dynamic clusters of T cell receptors at the T cell/antigen-presenting cell intercellular immune synapse and revealed the presence of numerous, highly dynamic long receptor-rich filopodial structures within inhibitory Natural Killer cell immune synapses.

V3 Spinal Neurons Establish a Robust and Balanced Locomotor Rhythm during Walking

A robust and well-organized rhythm is a key feature of many neuronal networks, including those that regulate essential behaviors such as circadian rhythmogenesis, breathing, and locomotion. Here we show that excitatory V3-derived neurons are necessary for a robust and organized locomotor rhythm during walking. When V3-mediated neurotransmission is selectively blocked by the expression of the tetanus toxin light chain subunit (TeNT), the regularity and robustness of the locomotor rhythm is severely perturbed. A similar degeneration in the locomotor rhythm occurs when the excitability of V3-derived neurons is reduced acutely by ligand-induced activation of the allatostatin receptor. The V3-derived neurons additionally function to balance the locomotor output between both halves of the spinal cord, thereby ensuring a symmetrical pattern of locomotor activity during walking. We propose that the V3 neurons establish a regular and balanced motor rhythm by distributing excitatory drive between both halves of the spinal cord.

Structural Basis for Pattern Recognition by the Receptor for Advanced Glycation End Products (RAGE)

The receptor for advanced glycated end products (RAGE) is a multiligand receptor that is implicated in the pathogenesis of various diseases, including diabetic complications, neurodegenerative disorders, and inflammatory responses. The ability of RAGE to recognize advanced glycated end products (AGEs) formed by nonenzymatic glycoxidation of cellular proteins places RAGE in the category of pattern recognition receptors. The structural mechanism of AGE recognition was an enigma due to the diversity of chemical structures found in AGE-modified proteins. Here, using NMR spectroscopy we showed that the immunoglobulin V-type domain of RAGE is responsible for recognizing various classes of AGEs. Three distinct surfaces of the V domain were identified to mediate AGE-V domain interactions. They are located in the positively charged areas of the V domain. The first interaction surface consists of strand C and loop CC ', the second interaction surface consists of strand C ', strand F, and loop FG, and the third interaction surface consists of strand A ' and loop EF. The secondary structure elements of the interaction surfaces exhibit significant flexibility on the ms-micros time scale. Despite highly specific AGE-V domain interactions, the binding affinity of AGEs for an isolated V domain is low, approximately 10 microm. Using in-cell fluorescence resonance energy transfer we show that RAGE is a constitutive oligomer on the plasma membrane. We propose that constitutive oligomerization of RAGE is responsible for recognizing patterns of AGE-modified proteins with affinities less than 100 nm.

Confocal microscopy applied to water mite taxonomy with the description of a new genus of Axonopsinae (Acari, Parasitengona, Hydrachnidia) from Central America

Vagabundia sci n. gen. n. sp. of the subfamily Axonopsinae is proposed and described. Confocal Laser Scanning Microscopy, not previously applied to water mite taxonomy, allowed the acquisition and posterior processing of clean optical slices. The new species is compared to other mites that have been described as ‘Axonopsella-like’. Vagabundia sci n. sp. is named after the Science Citation Index, a sociological tool that, as explained in the text, has done more harm than good to the population of taxonomists.

Index mapping for fibers with symmetric and asymmetric refractive index profiles

The application of phase imaging to refractive index profiling of an optical fiber slice is described. It is shown that the refractive index profile of axially symmetric and asymmetric optical fibers can be obtained from quantitative phase image of thin transverse optical fiber slices. Although this method requires careful and time consuming sample preparation, one advantage of this technique is that it can be applied to a wide range of optical fibers. In this paper results for both symmetric and non-symmetric fibers are presented and good agreement with the industry-standard refracted near-field technique demonstrated.

A New Branch of Endoplasmic Reticulum Stress Signaling and the Osmotic Signal Converge on Plant-specific Asparagine-rich Proteins to Promote Cell Death

NRPs (N-rich proteins) were identified as targets of a novel adaptive pathway that integrates endoplasmic reticulum (ER) and osmotic stress signals based on coordinate regulation and synergistic up-regulation by tunicamycin and polyethylene glycol treatments. This integrated pathway diverges from the molecular chaperone-inducing branch of the unfolded protein response (UPR) in several ways. While UPR-specific targets were inversely regulated by ER and osmotic stresses, NRPs required both signals for full activation. Furthermore, BiP (binding protein) overexpression in soybean prevented activation of the UPR by ER stress inducers, but did not affect activation of NRPs. We also found that this integrated pathway transduces a PCD signal generated by ER and osmotic stresses that result in the appearance of markers associated with leaf senescence. Overexpression of NRPs in soybean protoplasts induced caspase-3-like activity and promoted extensive DNA fragmentation. Furthermore, transient expression of NRPs in planta caused leaf yellowing, chlorophyll loss, malondialdehyde production, ethylene evolution, and induction of the senescence marker gene CP1. This phenotype was alleviated by the cytokinin zeatin, a potent senescence inhibitor. Collectively, these results indicate that ER stress induces leaf senescence through activation of plant-specific NRPs via a novel branch of the ER stress response.