Mark Kwaaitaal

Rik Huisman

Jens Maintz

Anja Reinstädler

Ralph Panstruga
email: panstruga@bio1.rwth-aachen.de

Ionotropic glutamate receptor (iGluR)-like channels mediate MAMP-induced calcium influx in Arabidopsis thaliana

Keywords: Calcium channel, calcium signature, elicitor, ionotropic glutamate receptor channel, microbeassociated molecular pattern, plant immunity

come

Introduction

Eukaryotic organisms possess an extensive array of receptor proteins to sense their environment, including the presence of benign or hostile (micro-) organisms. Major pathways for the relay of exogenous signals to the cytoplasm/nucleus invoke pattern recognition receptors (PRRs) that sense specific microbial epitopes termed microbe-associated molecular patterns (MAMPs). PRRs play a key role in plant innate immunity by the activation of defense response pathways upon MAMP sensing (MAMP-triggered immunity) [START_REF] Boller | A renaissance of elicitors: perception of microbeassociated molecular patterns and danger signals by pattern-recognition receptors[END_REF] Examples of PRRs comprise FLAGELLIN SENSING2 (FLS2), ELONGATION FACTOR-Thermo unstable (EF-Tu) RECEPTOR (EFR) and CHITIN ELICITOR RECEPTOR KINASE1 (CERK1). FLS2 and EFR activate downstream signaling after detection of 22 and 18 amino acid-long epitopes of bacterial flagellin (flg22) and EF-Tu (elf18), respectively [START_REF] Gómez-Gómez | FLS2: an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis[END_REF][START_REF] Zipfel | Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacteriummediated transformation[END_REF]. CERK1 is essential for the recognition of chitin polymers, major constituents of the fungal cell wall [START_REF] Miya | CERK1, a LysM receptor kinase, is essential for chitin elicitor signaling in Arabidopsis[END_REF]. Detection of MAMPs by PRRs typically results in a series of early cellular responses such as fluxes of calcium (Ca 2+ ) and other ions, the generation of reactive oxygen species (ROS), the initiation of mitogen-activated protein kinase cascades (MAPK(K/KK)) and the activation of Ca 2+ -dependent protein kinases (CDPKs) [START_REF] Boller | A renaissance of elicitors: perception of microbeassociated molecular patterns and danger signals by pattern-recognition receptors[END_REF]. Despite their canonical occurrence, the functional role of these archetypal responses in plant innate immunity remains largely obscure.

Ca 2+ acts as a second messenger in response to many biotic and abiotic stimuli, e.g. microbial attack, symbiosis, heat shock, cold shock, osmotic stress, oxidative stress, light conditions and abscisic acid [START_REF] Boller | A renaissance of elicitors: perception of microbeassociated molecular patterns and danger signals by pattern-recognition receptors[END_REF][START_REF] Lecourieux | Calcium in plant defence-signalling pathways[END_REF][START_REF] Aslam | Microbe-associated molecular pattern (MAMP) signatures, synergy, size and charge: influences on perception or mobility and host defence responses[END_REF]. These different cues each induce their own specific Ca 2+ signatures; transients that differ in timing as well as the number and magnitude of Ca 2+ spikes. The transient increase of cytosolic [Ca 2+ ] results from passive Ca 2+ influx from the apoplast or intracellular Ca 2+ stores through dedicated channels [START_REF] Bush | Calcium regulation in plant cells and its role in signaling[END_REF]. Since Ca 2+ is cytotoxic, low concentrations are maintained in the cytosol and nucleus of resting cells. H + /Ca 2+ -exchangers and ATP-dependent Ca 2+ pumps (Ca 2+ -ATPases) expel Ca 2+ in an energy-dependent process from the cytosol to the extracellular space and into intracellular compartments such as the vacuole and the endoplasmic reticulum (ER). As a result, the [Ca 2+ ] in the apoplast and intracellular stores is up to 20,000 times higher than in the cytosol. The combination of this concentration gradient and a proton-based electrochemical gradient over cellular membranes [START_REF] Mäser | Phylogenetic relationships within cation transporter families of Arabidopsis[END_REF] is the driving force for the stimulus-dependent influx of Ca 2+ into the cytosol [START_REF] Bush | Calcium regulation in plant cells and its role in signaling[END_REF].

Genome-wide comparisons revealed large differences between families for channel candidates mediating Ca 2+ import in plants and mammals [START_REF] Mäser | Phylogenetic relationships within cation transporter families of Arabidopsis[END_REF][START_REF] Wheeler | Ca 2+ signalling in plants and green algaechanging channels[END_REF][START_REF] Chiu | Phylogenetic and expression analysis of the glutamate-receptor-like gene family in Arabidopsis thaliana[END_REF]. Genes encoding obvious orthologs of some major classes of mammalian channels are lacking in plants (ryanodine receptor channels, InsP 3activated receptor channels and voltage-gated channels), while the family of cyclic nucleotide-gated channels (CNGCs) is expanded in plants. Even though sequence-related genes are missing, electrophysiological experiments indicated the presence of voltage-gated Ca 2+ channels in plant organelles and cells [START_REF] Demidchik | Arabidopsis thaliana root non-selective cation channels mediate calcium uptake and are involved in growth[END_REF]12]. Apparently, a unique and sequence-diverged set of proteins contributes to the control of Ca 2+ influx into plant cells.

CNGCs comprise a family of twenty ligand-gated ion channels in Arabidopsis thaliana [START_REF] Mäser | Phylogenetic relationships within cation transporter families of Arabidopsis[END_REF]. CNGCs are gated by cyclic nucleotides (cNTPs) [START_REF] Ma | Ca 2+ , cAMP, and transduction of non-self perception during plant immune responses[END_REF][START_REF] Qi | Ca 2+ signaling by plant Arabidopsis thaliana Pep peptides depends on AtPepR1, a receptor with guanylyl cyclase activity, and cGMP-activated Ca 2+ channels[END_REF]. Several CNGCs have been functionally linked to plant immunity. For example, the defense no death (dnd) class of mutants has a reduced ability to initiate a hypersensitive response (HR) to avirulent bacteria. DND1 and DND2 encode two CGNC paralogs (CNGC2 and CNGC4) [START_REF] Clough | The Arabidopsis dnd1 "defense, no death" gene encodes a mutated cyclic nucleotidegated ion channel[END_REF][START_REF] Jurkowski | Arabidopsis DND2, a second cyclic nucleotide-gated ion channel gene for which mutation causes the "defense, no death" phenotype[END_REF]. In addition to the altered cell death phenotype, cngc2 knockout plants show an altered Ca 2+ signature in response to the bacterial MAMP lipopolysaccharide (LPS) [START_REF] Ma | Ca 2+ , cAMP, and transduction of non-self perception during plant immune responses[END_REF].

The second major class of putative ligand-gated Ca 2+ -permeable channels in plants comprises the ionotropic glutamate receptor-like channels (iGluRs). They function as heterotetramers and are best known for their role in neurotransmission in vertebrates [START_REF] Tikhonov | Origin and molecular evolution of ionotropic glutamate receptors[END_REF]. iGluR-like channels possess a large N-terminal extracellular domain, three transmembrane regions, a hydrophobic loop defining the pore region and a cytosolic C-terminal domain. Especially the pore region of iGluR-like channels differs strongly between plants and animals [START_REF] Tapken | Arabidopsis thaliana glutamate receptor ion channel function demonstrated by ion pore transplantation[END_REF]19]. However, ion pore transplantation experiments have shown that the pores of two Arabidopsis iGluRs (AtiGluR1.1 and AtiGluR1.4) are permeable to Ca 2+ , Na + and K + [START_REF] Tapken | Arabidopsis thaliana glutamate receptor ion channel function demonstrated by ion pore transplantation[END_REF]. Twenty iGluRs are encoded in the Arabidopsis genome [START_REF] Wheeler | Ca 2+ signalling in plants and green algaechanging channels[END_REF][START_REF] Chiu | Phylogenetic and expression analysis of the glutamate-receptor-like gene family in Arabidopsis thaliana[END_REF]19]. They are involved in various processes ranging from biotic and abiotic stress responses [START_REF] Meyerhoff | AtGLR3.4, a glutamate receptor channel-like gene is sensitive to touch and cold[END_REF] to pollen tube Here we used a transgenic Arabidopsis thaliana line expressing the aequorin Ca 2+ biosensor to investigate intracellular Ca 2+ signatures in response to three different, well-characterized MAMPs (flg22, elf18 and chitin). We conducted pharmacological interference with the MAMP-triggered Ca 2+ influx using a comprehensive set of compounds known to impede various classes of Ca 2+ channels. Besides the actual Ca 2+ signatures, we used MAMP-induced downstream responses such as MAPK activation and accumulation of defense gene transcripts as additional readouts for Ca 2+ channel modulation. Our findings support a crucial role of iGluR-like channels in the generation of MAMPtriggered Ca 2+ signatures and provide evidence for a critical role for MAMP-induced Ca 2+ signaling in defense gene activation.

Experimental

Plant growth Arabidopsis Col-0 seeds or Col-0 35S::aequorin seeds [24] were surface-sterilized once with 70% ethanol and subsequently twice with 100% ethanol before drying. Dry seeds were transferred to a 96well plate (Perkin-Elmer, Waltham, USA; 1-2 seeds per well) containing 100 µl 1x MS including vitamins (DUCHEFA, Haarlem, the Netherlands) with 0,25% sucrose. The seedlings were grown for 11-12 days with 16 hours light (21 0 C), 8 hours dark (19 0 C) before further study.

Chemicals

Unless stated otherwise, chemicals were obtained from SIGMA (Munich, Germany) or Tocris Biosciences (Ellisville, USA). Stock solutions were prepared in ddH 2 O except for CNQX, U73122 and nifedipine, which were dissolved in DMSO. The influence of the solvent DMSO in the final concentration after chemical dilution was tested as an additional control.

Ca 2+ measurements using the aequorin biosensor

After 11-12 days the growth medium was removed from the aequorin expressing seedlings and replaced by 100 µl ddH 2 0 containing 10 µM coelenterazine (BIOSYNTH, Staad, Switzerland), diluted from a 5 mM stock solution in methanol, and incubated overnight in the dark. The next day, the medium was replaced by 100 µl ddH 2 0 and the seedlings were incubated for 30 min in the dark. For the compound treatments, 10-times concentrated stocks in water were prepared and added to the coelenterazine-loaded seedlings. The seedlings were preincubated for one hour before starting the measurements. Elf18 and flg22 were present as 10 or 1 mM stock solutions in water stored at -80 0 C and used freshly. Chitin solution was prepared by intermittently grinding (for 15-20 minutes) 10 mg crab or shrimp shell (SIGMA, Munich, Germany) powder in 1 ml ddH 2 0 with a pestle and subsequent vortexing and heating the solution to 60 0 C. Undissolved particles were removed by centrifugation (15 minutes, 13000 rpm) and the clear supernatant was used for the experiments. The measurements were performed in a 96-well Centro LB960 luminometer system (Berthold Technologies, Bad Wildbad, Germany). Half a plate was measured in one go. Luminescence from one single well was detected for 0,25 seconds and each well was measured every 30 seconds. After 2 minutes (4 measurements), 5-times concentrated MAMP was automatically added to a final concentration of 1 µM flg22, 1 µM elf18 or 0,1 mg/ml chitin. After addition, the luminescence was followed for an additional 40 minutes. The second half of the plate was run in the same manner. Finally, for calculation of Ca 2+ concentrations, 100 µl 2 M CaCl 2 in 20% ethanol was added to each well using the injectors of the system and the luminescence was measured for 0,25s per well with one measurement per well every 63 seconds. The luminescence was followed for 30 minutes. Ca 2+ concentrations were calculated according to [START_REF] Rentel | Oxidative stress-induced calcium signaling in Arabidopsis[END_REF] [START_REF] Rentel | Oxidative stress-induced calcium signaling in Arabidopsis[END_REF].

Per treatment 4 to 6 individual wells were averaged. Ca 2+ transients were compared between treatments within one experiment unless stated otherwise and at least three biological replicates per compound were performed. Unless stated otherwise, the data were normalized to background Ca 2+ levels and represented as a Δ[Ca 2+ ] value to compensate for background variations between measurements. Owing to the low molecular mass of aequorin (22 kDa) and its presumed passive transfer into the nucleus, we expect the Arabidopsis reporter line to monitor the combined cytoplasmic and nuclear (here referred to as intracellular) Ca 2+ pool. Statistical significance of differences was evaluated by comparing average calcium responses of biological replicates of treated samples with non-treated control samples using a two-sided Student's t-test with unequal variances.

MAPK assays

The MAPK assays were performed essentially as previously described [START_REF] Saijo | Receptor quality control in the endoplasmic reticulum for plant innate immunity[END_REF].

Quantitative RT-PCR Preparation, pretreatment and MAMP addition to the seedlings was identical as described for the MAPK assays. Time point 0 was harvested before MAMP addition and 30 and 60 minutes after MAMP addition. Total RNA was isolated using the RNeasy Plant Kit (QIAGEN, Hilden, Germany). cDNA was synthesized with 5 µg total RNA, oligo dT primers and Superscript II reverse transcriptase (Invitrogen, Karlsruhe, Germany) according to manufacturers protocol. Transcript abundance was quantified using the IQ5 real-time PCR thermocycler (Bio-Rad, Munich, Germany). The expression of the genes of interest was normalized to At4g26410 (for primers used see [START_REF] Czechowski | Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis[END_REF]) and to the relative transcript abundance in non-treated Col-0 control samples as calculated according to the comparative cycle threshold (∆∆Ct) method [28]. Transcript abundance of a MAPK dependent gene, FRK1 and a CDPK dependent gene, PHI-1, were tested (for primers used see [29]). Three biological replicates with three technical replicates per sample were performed per treatment.

Results

MAMP-triggered Ca 2+ influx from the apoplast

We used the luminescent aequorin Ca 2+ sensor system [24] to record whole seedling intracellular Ca 2+ transients in response to MAMPs. Owing to previous in-depth characterization and the identification of the cognate PRRs we focused on responses towards the bacterial peptide elicitors flg22 and elf18 [START_REF] Gómez-Gómez | FLS2: an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis[END_REF][START_REF] Zipfel | Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacteriummediated transformation[END_REF] as well as chitin, a carbohydrate polymer that represents the major constituent of fungal cell walls [START_REF] Felix | Specific perception of subnanomolar concentrations of chitin fragments by tomato cells: induction of extracellular alkalinization, changes in protein-phosphorylation, and establishment of a refractory state[END_REF]. To narrow down candidate protein classes involved in regulating MAMP-triggered Ca 2+ signatures in Arabidopsis, a range of chemicals were chosen that are known to target components controlling Ca 2+ in-and efflux in plant and animal systems. Due to potential plant off-targets, presumed differences in bioavailability, compound metabolism and dissimilarities in the molecular structure of plant and animal targets we generally tested multiple chemicals affecting the same molecule class.

To investigate the source of the initial intracellular [Ca 2+ ] rise, the general Ca 2+ channel inhibitor Lanthanum chloride (LaCl 3 ) and a chelator of extracellular bivalent cations, ethylene glycolbis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), were applied. Consistent with previous reports [START_REF] Aslam | Microbe-associated molecular pattern (MAMP) signatures, synergy, size and charge: influences on perception or mobility and host defence responses[END_REF]31] flg22 and elf18 induced a biphasic Ca 2+ response with a maximum [Ca 2+ ] at about three minutes after MAMP addition and a recovery phase of ca. 20-30 minutes (Figures 1A and1B). Chitin induced a monophasic Ca 2+ transient (Figure 1C). Preincubation of the seedlings with either LaCl 3 or EGTA fully inhibited the Ca 2+ transients induced by all three MAMPs, suggesting the main Ca 2+ source for the MAMP-triggered spikes is the apoplast (Figures 1, A-D, Supplemental Figures S1A andB and Table 1). In the presence of EGTA, a continuous rise in intracellular Ca 2+ levels was observed, independent of the presence or absence of MAMPs (Figure 1D, Supplemental Figure S1A andB).

An ambiguous role for internal Ca 2+ stores in MAMP-triggered Ca 2+ influx

To further validate the conclusion that the major source of MAMP-induced Ca 2+ is the apoplast we studied the role of phospholipase C (PLC)-dependent release of Ca 2+ from intracellular stores using the PLC inhibitors neomycin and U73122 [START_REF] De Jong | Phosphatidic acid accumulation is an early response in the Cf-4/Avr4 interaction[END_REF][START_REF] Munnik | Plant phospholipid signaling: "in a nutshell[END_REF]. Both inhibitors led to increased steady state Ca 2+ levels (Figure 2A, Supplemental Figure S2C and Table 1). U73122 did not alter Δ[Ca 2+ ] for the tested MAMPs, but changed the shape of the response curves of elf18 and chitin (Figure 2B, Supplemental Figures S2A andB), which suggests a minor contribution for PLC-controlled Ca 2+ release in response to these MAMPs. Neomycin did not alter the chitin-induced Δ[Ca 2+ ] (Supplemental Figure S2F), but caused a slight reduction in the flg22-and elf18-induced Δ[Ca 2+ ] (Supplemental Figures S2C-E). In conclusion, PLC-mediated Ca 2+ release has a minor, if any, contribution to the MAMP-triggered Δ[Ca 2+ ].

A MAMP-triggered primary apoplastic Ca 2+ influx does not exclude the possibility of secondary Ca 2+ -induced Ca 2+ release (CICR) from internal stores. Ruthenium Red (RR) inhibits CICR from internal stores in plant and animal systems [START_REF] Endo | Calcium-induced calcium release in skeletal muscle[END_REF], but also inhibits certain channels at the plasma membrane [START_REF] Pineros | Calcium channels in higher plant cells: selectivity, regulation and pharmacology[END_REF][START_REF] Pottosin | Cooperative block of the plant endomembrane ion channel by ruthenium red[END_REF]. Like inhibition of the PLC pathway, addition of RR resulted in increased steady state [Ca 2+ ] (Supplemental Figure S2G and Table 1). In addition, the chitin-and elf18-triggered Δ[Ca 2+ ] were strongly reduced compared to the uninhibited situation (Figures 2C andD), while the flg22-induced Δ[Ca 2+ ] was largely unaffected (Supplemental Figures S2G andH). This suggests either a significant contribution of CICR from intracellular stores or, alternatively, a role for specific RRtargeted plasma membrane-localized channels in the chitin and elf18 responses. Like RR, tetracaine directly targets mammalian ryanodine receptors and blocks CICR from intracellular stores [START_REF] Endo | Calcium-induced calcium release in skeletal muscle[END_REF]. Tetracaine induced a higher Δ[Ca 2+ ] for all three MAMPs and interfered with the recovery phase, visible as irregular spikes during the decline of the Ca 2+ response (Figure 2E, Supplemental Figures S2I-J and Table 1). Unlike for the other intracellular store channel inhibitors, in the case of tetracaine no increased steady state [Ca 2+ ] was observed (not shown). In sum, the compounds targeting internal Ca 2+ stores yielded ambiguous data. A role for PLC-dependent Ca 2+ release and /or CICR in response to MAMP treatment can thus not be ruled out.

Inhibitors of voltage-and nucleotide-gated channels do not affect MAMP-triggered Ca 2+ influx

To further delimit the channels involved in Ca 2+ import from extracellular stores, a set of inhibitors targeting animal type voltage-gated Ca 2+ channels (VGCC) were tested. Although plants lack obvious candidate genes coding for VGCCs, electrophysiological data support their existence in plant organelles and cells [START_REF] Demidchik | Arabidopsis thaliana root non-selective cation channels mediate calcium uptake and are involved in growth[END_REF]12]. None of the three L-type VGCC inhibitors, verapamil, diltiazem and nifedipine, altered the Δ[Ca 2+ ] after MAMP stimulus (Figure 2F and Supplemental Figures S3A-H and Table 1). Nifedipine treatment resulted in a delayed recovery in the case of all three MAMPs (Figure 2F and Supplemental Figures S3A-B and Table 1). Verapamil-treated seedlings occasionally showed random spiking after the MAMP stimulus (Supplemental Figures S3C-E and Table 1). Together, these results suggest that the sensing of flg22, elf18 or chitin does not activate Ca 2+ influx through VGCCs.

Plant genomes encode two major classes of putative Ca 2+ channels, CNGCs and iGluRs. Two compounds were tested that interfere with cNTP biosynthesis and therefore indirectly inhibit CNGC function, alloxan and dideoxyadenosine (DDA). Neither incubation of seedlings with alloxan nor treatment with DDA caused an alteration of the MAMP-induced Δ[Ca 2+ ] for flg22 (Figures 3A andB, Supplemental Figure S5C and Table 1). For elf18 and chitin the Δ[Ca 2+ ] was slightly lower in the presence of alloxan or DDA (Supplemental Figures S4A-D, Supplemental Figures S5F andI and Table 1) DDA pretreatment resulted in a delayed recovery of cellular Ca 2+ levels after MAMP stimulation (Figure 3B, Supplemental Figures S4C andD and Table 1). These results suggest that, dissimilar to lipopolysaccharides (LPS) [START_REF] Ma | Ca 2+ , cAMP, and transduction of non-self perception during plant immune responses[END_REF] cyclic nucleotide biosynthesis and thus likely also CNGCs do not make a major contribution to the flg22-, elf18-or chitin-induced Ca 2+ influx.

Inhibition of iGluR-like channels affects MAMP-triggered Ca 2+ influx

The second major class of putative Ca 2+ channels in plants comprises the iGluR-like channels, which include 20 members in Arabidopsis [START_REF] Wheeler | Ca 2+ signalling in plants and green algaechanging channels[END_REF][START_REF] Chiu | Phylogenetic and expression analysis of the glutamate-receptor-like gene family in Arabidopsis thaliana[END_REF]19]. We employed various inhibitors directed against NMDA-and non-NMDA-like mammalian iGluRs. AP-5 and AP-7, competitive antagonists of the glutamate binding site [START_REF] Foster | The novel anticonvulsant MK-801 binds to the activated state of the N-methyl-D-aspartate receptor in rat brain[END_REF] and kynurenic acid, a competitive antagonist of the glycine binding site [START_REF] Bertolino | Kynurenic acid inhibits the activation of kainic and N-methyl-D-aspartic acid-sensitive ionotropic receptors by a different mechanism[END_REF], caused a strong inhibition of the MAMP-triggered Δ[Ca 2+ ]. For all three MAMPs, the reduction induced by both AP-5 and kynurenic acid was statistically significant (p<0,05). Treatment with these inhibitors also led to increased steady state [Ca 2+ ] (Figures 4A-C, Supplemental Figures S4E-H and Table 1). The observation that increased cellular Ca 2+ concentrations in the presence of U73122 still allowed a normal flg22-, elf18-and chitin-induced ∆[Ca 2+ ] (Figures 2A andB, Supplemental Figures S2A andB) suggests that elevated steady state Ca 2+ levels do not per se impede a MAMP-triggered Ca 2+ transient. Other iGluR channel inhibitors tested targeting either the channel domain of NMDAlike channels (MK-801) [START_REF] Foster | The novel anticonvulsant MK-801 binds to the activated state of the N-methyl-D-aspartate receptor in rat brain[END_REF] or non-NMDA iGluR channels (DNQX, CNQX) [START_REF] Honore | Quinoxalinediones: potent competitive non-NMDA glutamate receptor antagonists[END_REF] did not inhibit MAMP-triggered Δ[Ca 2+ ] (Supplemental Figure S6 and Table 1). Together, these data provide first evidence for a potential role of iGluR-like channels in the generation of the flg22-, elf18-and chitintriggered Ca 2+ transient.

To further analyze the potential role of iGluRs in MAMP-induced Ca 2+ influx, we exploited the phenomenon of iGluR desensitization [START_REF] Stephens | Glutamate receptor subtypes evidenced by differences in desensitization and dependence on the GLR3.3 and GLR3.4 genes[END_REF]. If iGluRs were the channels mediating MAMPtriggered Ca 2+ influx, then agonistic amino acids such as L-glutamate and L-aspartate should desensitize the channels for subsequent MAMP treatment, while other amino acids should have either less or no effect. We thus monitored the MAMP-triggered Ca 2+ response after preincubation of the aequorin-expressing seedlings with ten different amino acids that differ largely in their physiochemical properties (Figure 4F). Pretreatment of seedlings with both L-glutamate and L-aspartate resulted in a statistically significant reduction (p<0,05) of the Δ[Ca 2+ ] after flg22 stimulation (Figures 4D-E, Supplemental Figures S4I-J and Table 1). In addition, reminiscent of treatment with the inhibitors AP-5, AP-7 and kynurenic acid, basal cellular [Ca 2+ ] levels were elevated to 125%-158% of the control following application of these two negatively charged amino acids (Figure 4D and F and Table 1). Other amino acids did neither reduce the MAMP-induced Δ[Ca 2+ ] nor affect basal cellular [Ca 2+ ] (Figure 4F).

We then tested a range of L-glutamate concentrations for desensitization of the flg22-mediated Ca 2+ transient and established a dose-response curve (Figure 4G). We found a concentrationdependent reduction of Δ[Ca 2+ ] and a reciprocal concentration-dependent increase in steady state Ca 2+ levels. From a concentration of 0,2 mM L-glutamate onwards a reduction of ∆[Ca 2+ ] and an increase in basal cellular [Ca 2+ ] was observed. Pretreatment with concentrations above 0,6 mM L-glutamate resulted in maximal inhibition of the flg22-triggered Ca 2+ transient, indicating saturation of the system (Figure 4G). The concentration range where L-glutamate desensitized MAMP-induced Ca 2+ influx is in agreement with the reported K m values for plant iGluR-like channels, which were found to be between 0,2 and 0,5 mM [START_REF] Demidchik | Glutamate activates cation currents in the plasma membrane of Arabidopsis root cells[END_REF], similar to reported apoplastic amino acid concentrations (0,3-1,3 mM) [42,[START_REF] Forde | Glutamate in plants: metabolism, regulation, and signalling[END_REF]. In sum, the results of desensitization experiments further support a role of iGluRs in MAMPtriggered Ca 2+ influx.

iGluR inhibitors and L-glutamate modulate MAMP-induced MAPK activation

To expand the analysis of cellular responses induced by compounds that affect iGluR activity, we inspected other early MAMP-triggered events. Upon biotic stress specific MAPK signaling cascades are activated, which control additional cellular responses and defense gene expression [START_REF] Pitzschke | MAPK cascade signalling networks in plant defence[END_REF]. We examined MAMP-induced MAPK activation in the presence or absence of L-glutamate or the iGluR inhibitors kynurenic acid or CNQX. Application of L-glutamate as a stimulus to seedlings resulted in low levels of MAPK activation, visible as three additional bands (MPK6, MPK3 and MPK4) appearing at five minutes after addition of L-glutamate or the MAMP stimulus (Figure 5A). Preincubation with either L-glutamate or kynurenic acid for one hour resulted in low levels of MAPK activation. Subsequent addition of more L-glutamate still transiently increased activated MAPK levels. Preincubation of the seedlings with CNQX, which did not affect the MAMP-triggered Ca 2+ signature, completely abolished MAPK activation after L-glutamate addition (Figure 5A).

Flg22, elf18 and chitin induced strong MAPK activation within 5 minutes after MAMP addition (Figures 5B andC and Supplemental Figure S7). In the presence of L-glutamate, kynurenic acid or CNQX, the flg22 and elf18 response was only affected to a minor extent. The presence of Lglutamate or kynurenic acid often delayed MAPK activation, with full activation not before 15 minutes (Supplemental Figure S7). In contrast, all three compounds markedly reduced the chitintriggered activation of the MAPK(K/KK) cascade (Figure 5B).

Recently, Boudsocq and co-workers [29] showed that flg22-induced MAPK activation in Arabidopsis protoplasts does not depend, but is enhanced by Ca 2+ influx. To corroborate this observation in intact Arabidopsis seedlings and to compare it with the glutamate-dependent inhibition of MAPK activation, we performed MAPK assays in the presence of either EGTA or LaCl 3 . In the case of flg22, full MAPK activation was delayed by both EGTA and LaCl 3 (Supplemental Figure S7B). In the case of elf18 and chitin, EGTA reduced and LaCl 3 delayed MAPK activation (Figure 5C and Supplemental Figure S7D). In conclusion, MAMP-triggered MAPK signaling requires Ca 2+ influx for full and timely activation. Modulation of MAMP-triggered Ca 2+ influx by iGluR inhibitors or glutamate desensitization affected MAPK activation in a similar manner as full inhibition (Figures 1A-D) of apoplastic Ca 2+ influx by the potent non-selective chemicals LaCl 3 and EGTA.

Cooperative regulation of Ca 2+ influx by CNGC and iGluR-like channels?

Modulating glutamate-controlled Ca 2+ influx did largely but not fully abolish the MAMP-triggered Ca 2+ transient (Figure 4; Supplemental Figures S4 andS5). Therefore we wondered if cNTP-regulated Ca 2+ import was responsible for the remaining influx of Ca 2+ and possibly a positive feedback loop exists between both channel types. Plants were co-treated with both kynurenic acid/L-glutamate and alloxan/dideoxyadenosine before MAMP addition and both Ca 2+ levels and MAPK activation were followed. For elf18, but not for flg22 and chitin, the combined incubations gave a stronger reduction in Δ[Ca 2+ ] than each of the single compounds (compare Figure 6A with Supplemental Figures S8A andC, Table 1). As discussed above, L-glutamate pretreatment reduced (chitin) or delayed (flg22 and elf18) MAPK activation. Alloxan pretreatment did not influence the intensity of the two major MAPK bands (MPK6 and MPK3), but reduced the intensity of the MAPK band with the highest electrophoretic mobility, which likely represents MPK4 (Figure 6B and Supplemental Figures S8B andD). The co-inhibition of cNTP-and iGluR-mediated Ca 2+ influx resulted in a combination of both effects; the major MAPK bands representing MPK3 and 6 showed a delayed activation (elf18) or reduced intensity (chitin) and nearly no activation of MPK4 could be detected anymore (Figure 6B and Supplemental Figures S8B, D andF). In conclusion, we did not find evidence for a strong cooperative effect of iGluR-like channels and CNGCs in MAMP-triggered Ca 2+ influx.

Glutamate signaling differentially modulates MAPK-and CDPK-dependent defense gene transcript levels in response to MAMPs

To investigate how the modulation of iGluR-like channels impacts on MAMP-triggered transcriptional activation of defense genes, we studied transcript accumulation of each a MAPK-and a CDPKdependent gene (FLG22-INDUCED-RECEPTOR-LIKE-KINASE1, FRK1, and PHOSPHATE-INDUCED1, PHI-1, respectively) [29] using quantitative RT-PCR. FRK1 transcript accumulation was rapidly (within 30 minutes) induced by each of the three MAMPs (Figure 7A). Application of either L-glutamate or kynurenic acid reduced basal FRK1 expression levels and drastically impaired MAMPdependent accumulation of FRK1 transcripts (Figure 7A). PHI-1 transcript levels were strongly and transiently induced by flg22 and moderately by elf18 or chitin (Figure 7B). L-glutamate and kynurenic acid each increased basal PHI-1 expression levels and blocked an additional increase in transcript accumulation upon MAMP treatment. Thus, modulation of MAMP-triggered Ca 2+ influx by Lglutamate or kynurenic acid inhibits transcriptional up-regulation of genes downstream of the MAPK (FRK1) and CDPK (PHI-1) signaling cascades.

Discussion

We tested a comprehensive set of compounds known to target various components regulating Ca 2+ signaling for their influence on MAMP-induced Ca 2+ influx in Arabidopsis seedlings. Pharmacological approaches bear the risk of unspecific off-targets, but offer the advantage to overcome potential redundancy in target function and allow transient inhibition with minimal pleiotropic effects. We opted for a pharmacological approach to surmount the presumed genetic redundancy of plant Ca 2+ channels and the expected pleiotropic phenotypes of respective mutants. We aimed to minimize the impact of off-targets on our data by applying multiple inhibitors per target molecule class in most cases.

Inhibition of iGluR-like channels affects MAMP-triggered Ca 2+ influx

Flg22 and elf18 induced biphasic Ca 2+ transients that were very similar in shape and timing, while chitin caused a monophasic Ca 2+ transient (e.g. Figure 1A andC). The presence of two separate maxima in the case of flg22 and elf18 suggests either the involvement of different channel types, different subcellular Ca 2+ -stores or a differential responsiveness between cell types. The aequorin reporter system used in this study does not allow the detection of changes in [Ca 2+ ] at the (sub)cellular level. Therefore, other reporters like the yellow cameleon FRET sensor are desirable for future analyses with improved spatial resolution [START_REF] Nagai | Expanded dynamic range of fluorescent indicators for Ca 2+ by circularly permuted yellow fluorescent proteins[END_REF][START_REF] Monshausen | Imaging of the yellow cameleon 3.6 indicator reveals that elevations in cytosolic Ca 2+ follow oscillating increases in growth in root hairs of Arabidopsis[END_REF]. LaCl 3 -mediated inhibition of plasma membrane-resident ion channels and chelating external Ca 2+ via EGTA treatment revealed that the primary Ca 2+ pool for both types of MAMP-triggered Ca 2+signatures is the apoplast (Figures 1A-D and Supplemental Figures S1A-C). This confirms and extends findings of a previous study [START_REF] Jeworutzki | Early signaling through the Arabidopsis pattern recognition receptors FLS2 and EFR involves Ca 2+ -associated opening of plasma membrane anion channels[END_REF], where LaCl 3 was used to demonstrate that flg22-induced Ca 2+ influx is needed for membrane depolarization in mesophyll cells. Furthermore, both inhibition of Ca 2+ influx by LaCl 3 and chelating apoplastic Ca 2+ were shown to prevent CDPK activation and to reduce MAPK activation in response to the peptide elicitors flg22 and elf18 in Arabidopsis protoplasts [29]. These findings suggest a role for Ca 2+ signaling in modulating downstream responses during MAMP-triggered immunity.

Interference with InsP 3 or CICR resulted in increased steady state Ca 2+ levels for three of the inhibitors tested (U73122, Neomycin and RR). Notably, although basal Ca 2+ levels were increased, a normal Ca 2+ transient was induced (Figures 2A-B). In addition, RR reduced the chitin-and elf18induced Ca 2+ transients, possibly due its inhibitory effect on plasma membrane-resident ion channels [START_REF] Pineros | Calcium channels in higher plant cells: selectivity, regulation and pharmacology[END_REF][START_REF] Pottosin | Cooperative block of the plant endomembrane ion channel by ruthenium red[END_REF]. The fourth compound, tetracaine, caused a slightly enhanced Ca 2+ transient, but did not alter basal Ca 2+ levels.

Inhibition of VGCCs did not change the intensity of MAMP-triggered Ca 2+ transients, but for two inhibitors (verapamil and nifedipine) delayed the recovery to basal cytosolic Ca 2+ levels (Figure 2F and Supplemental Figure S3). VGCC inhibitors have documented effects in plants [START_REF] Pineros | Calcium channels in higher plant cells: selectivity, regulation and pharmacology[END_REF]48]. However, all three L-type channel inhibitors have also other reported targets than L-type Ca 2+ channels, including sodium channels and membrane receptors (https://www.ebi.ac.uk/chembldb/). The prolonged Ca 2+ signature might thus be due to interference with other ion channel types and a resulting altered recovery of the membrane potential or ion gradients including Ca 2+ .

Inhibitors of CNGCs previously shown to inhibit CNGC-mediated responses in planta [START_REF] Ma | Ca 2+ , cAMP, and transduction of non-self perception during plant immune responses[END_REF], DDA and alloxan, caused only a slight reduction in the MAMP-triggered Ca 2+ transients. Therefore in contrast to LPS [START_REF] Ma | Ca 2+ , cAMP, and transduction of non-self perception during plant immune responses[END_REF] and the Danger-Associated Molecular Pattern (DAMP) AtPep3 [START_REF] Qi | Ca 2+ signaling by plant Arabidopsis thaliana Pep peptides depends on AtPepR1, a receptor with guanylyl cyclase activity, and cGMP-activated Ca 2+ channels[END_REF], cNTPs and thus likely CNGCs do not control the flg22-, elf18-and chitin-induced Ca 2+ transient. cngc2 mutants lose the LPS-induced Ca 2+ increase [START_REF] Ma | Ca 2+ , cAMP, and transduction of non-self perception during plant immune responses[END_REF], but show normal flg22-triggered membrane depolarization [START_REF] Jeworutzki | Early signaling through the Arabidopsis pattern recognition receptors FLS2 and EFR involves Ca 2+ -associated opening of plasma membrane anion channels[END_REF]. It appears that the MAMPs used in the present study activate a different set of ion channels than LPS does. The proposed discrepancy between the channels involved is in line with the different gene sets activated by the two groups of elicitors [29]. Surprisingly, the flg22-induced Ca 2+ transient is attenuated in a Atpepr1 mutant background [START_REF] Qi | Ca 2+ signaling by plant Arabidopsis thaliana Pep peptides depends on AtPepR1, a receptor with guanylyl cyclase activity, and cGMP-activated Ca 2+ channels[END_REF], which suggests crosstalk at the level of Ca 2+ influx between the AtPep and flg22/elf18/chitin pathways independent of cNTP generation. The delayed recovery of the MAMP-induced cytosolic rise in Ca 2+ levels in the presence of DDA suggests a role for cNTPs in controlling Ca 2+ efflux.

Of the six iGluR inhibitors tested, AP-5, AP-7 and kynurenic acid resulted in a strong reduction in the MAMP-induced ∆[Ca 2+ ] and all three led to an increase in cytosolic [Ca 2+ ] (Figures 4A-C , Supplemental Figures S4-6 and Table 1 and2). MK-801, CNQX and DNQX pretreatment did not alter flg22-, elf18-or chitin-induced Ca 2+ fluxes (Supplemental Figure S6). AP-5, CNQX and DNQX have been previously shown to affect iGluR-mediated processes and Ca 2+ currents in pollen tube growth (AP-5, CNQX, DNQX) [START_REF] Michard | Glutamate receptor-like genes form Ca 2+ channels in pollen tubes and are regulated by pistil D-serine[END_REF], hypocotyl elongation (DNQX)[22], glutamate-induced Ca 2+ transients in mesophyll cells (CNQX, DNQX) [START_REF] Meyerhoff | AtGLR3.4, a glutamate receptor channel-like gene is sensitive to touch and cold[END_REF] and responses to Al 3+ -induced Ca 2+ transients in roots (AP-5) [48]. Most recently, AP-5 was found to strongly inhibit Ca 2+ influx induced by the peptide elicitor cryptogein in tobacco cell culture, while MK-801 and DNQX were significantly less effective in these experiments [23]. Based on these published findings, a subset of the iGluR inhibitors can efficiently modulate both NMDA-and non-NMDA-like iGluR channels. In our hands, DNQX, CNQX and MK-801 did not result in inhibition of MAMP-induced Ca 2+ influx (Supplemental Figure S6). Possibly these compounds were unstable in our experimental conditions or were unable to penetrate the seedling tissue well enough to compete with glutamate for binding sites. In pollen tubes, AP-5 had a stronger influence on Ca 2+ currents than CNQX and DNQX, suggesting that higher apoplastic concentrations of the latter compounds are needed [START_REF] Michard | Glutamate receptor-like genes form Ca 2+ channels in pollen tubes and are regulated by pistil D-serine[END_REF], which might not have been achieved in our experimental set-up. Alternatively, various plant iGluR channels or iGluR channel complexes may differ in their sensitivity towards the inhibitors. The channel domain of some plant iGluR-like channels are permeable to Ca 2+ , Na + and K + [START_REF] Tapken | Arabidopsis thaliana glutamate receptor ion channel function demonstrated by ion pore transplantation[END_REF], but they differ in conserved amino acids controlling channel selectivity from animal iGluRs [START_REF] Tapken | Arabidopsis thaliana glutamate receptor ion channel function demonstrated by ion pore transplantation[END_REF]19], which might explain the inactivity of MK-801 in our experiments. Interestingly, inhibitor treatment and de-sensitization of the MAMP responses with L-glutamate or L-aspartate resulted in similar changes of the Ca 2+ response and increased steady state [Ca 2+ ] (Figure 4, Table 1 and2). The increased steady state Ca 2+ levels in the presence of both inhibitors and agonists can be best rationalized by either an unexpected partial agonistic function of the presumed inhibitors or by hyper-compensation of interference with iGluR-like channel function via other Ca 2+ channel types.

Inhibition of iGluR-like channel does not fully abolish the Ca 2+ spike, which suggests either partial inhibition of these channels or the involvement of a second channel class in the response. A second channel class acting in the flg22-and elf18-mediated Ca 2+ influx would explain the biphasic shape of the Ca 2+ response for these MAMPs. However, chitin-induced fluxes are altered in a similar manner, which argues for an alternative explanation of the phenomenon. Our experiments suggest the potential second class of channels is not cNTP-activated, since co-treatment with inhibitors of adenylate cyclases and iGluRs did not fully block Ca 2+ influx (Figure 6A).

We exploited the phenomenon of iGluR channel desensitization by analyzing the MAMP response after pretreatment with various amino acids. In our experiments, flg22-, elf18-and chitininduced responses were only desensitized by the negatively charged L-glutamate and L-aspartate (Figures 4D-G, Supplemental Figures S4I andJ). In a previous study certain combinations of amino acids were tested for cross-desensitization and the dependence on the presence of AtGLR3.3 and AtGLR3.4 [START_REF] Stephens | Glutamate receptor subtypes evidenced by differences in desensitization and dependence on the GLR3.3 and GLR3.4 genes[END_REF]. The results of this work fit a model where various quaternary complexes of iGluRlike channels co-exist that vary in their sensitivity to different amino acids. Taking this model into account, the iGluR complex(es) activated by MAMPs only seem(s) to contain subunits sensitive to both L-glutamate and L-aspartate. Comparative modeling of the glutamate binding site of plant iGluRlike channels suggested that only AtGLR1.1 can bind glutamate, while the other family members bind glycine [22]. This suggests that the iGluR complex responsible for MAMP-triggered Ca 2+ influx would have to contain AtGLR1.1. However, modeling studies alone are insufficient to exclude binding of other amino acids [START_REF] Qi | Calcium entry mediated by GLR3.3, an Arabidopsis glutamate receptor with a broad agonist profile[END_REF]. Furthermore, L-glutamate or L-aspartate might not directly bind the receptor, but induce the release of another compound that modulates iGluR-like channel activity [START_REF] Qi | Calcium entry mediated by GLR3.3, an Arabidopsis glutamate receptor with a broad agonist profile[END_REF].

iGluR-mediated Ca 2+ influx controls defense gene expression

Reduction of MAMP-triggered Ca 2+ influx fully inhibited expression of a MAPK-dependent gene (FRK1) and increased steady state levels of a CDPK-dependent gene (PHI-1) (Figure 7) [29]. Given the only modest effect of L-glutamate und kynurenic acid on MAPK activation (Figure 5 and Supplemental Figure S7) the strong impact on MAPK-dependent FRK1 expression was surprising. This result suggests that either the signal transduction cascade leading from Ca 2+ influx to the activation of FRK1 transcript accumulation relies only partially on MAPK activation, or that a moderate impact on MAPK activity can have a profound effect on downstream gene expression. The enhanced steady state PHI-1 transcript levels seen upon treatment with L-glutamate or kynurenic acid are possibly a consequence of the enhanced intracellular steady state Ca 2+ levels caused by these compounds, which may lead to constitutive low level activation of CDPKs. Notably, a further MAMPinduced increase in PHI-1 transcript levels was also blocked by L-glutamate and kynurenic acid. In sum, these data indicate a crucial role for cellular signaling events upon MAMP perception that rely on iGluR-mediated Ca 2+ influx. It remains to be seen to what extent the reduction in defense gene activation translates into a compromised immune response. In this context it would be highly desirable to examine the effect of interference with Ca 2+ signaling during authentic plant-microbe interactions. However, the dual effect of compounds on both the host and the parasite combined with the resulting complication in interpreting the data from such experiments emphasize the limitations of pharmacological approaches.

A model for iGluR-mediated Ca 2+ influx

Based on our data we propose the following model for MAMP-triggered Ca 2+ influx (Figure 8): Following MAMP perception by cognate PRRs, glutamate-dependent opening of iGluR-like channels and Ca 2+ influx from the apoplast into the cytoplasm is activated by yet unknown mechanisms. This may involve vesicle-mediated secretion of L-glutamate into the apoplast [23]. Elevated cytoplasmic Ca 2+ levels translate via CDPK-and MAPK-dependent pathways into defense gene activation. It remains unclear how Ca 2+ influx via iGluRs is terminated. One possibility is a negative feedback loop that involves Ca 2+ -dependent calmodulin (CaM) binding to hypothetical CaM binding domain(s) of iGluR-like channels. As a next step, reverse genetic approaches are required to further substantiate the findings of the present study at a different experimental level. In our hands the analysis of a subset of iGluR single mutants did not yet result in the identification of mutants that exhibit altered MAMPinduced Ca 2+ signatures. These preliminary data indeed argue for redundant iGluR functions in plant innate immunity.

Figure legends

Figure 1 flg22, elf18 and chitin induce Ca 2+ influx from the apoplast. 10 to 12-day-old aequorin-expressing Arabidopsis seedlings were pretreated for one hour with 1 mM LaCl 3 (A-C) or 10 mM EGTA (D). The measurements were started and after two minutes, flg22 (1 µM, A and D), elf18 (1 µM, B) or chitin (0,1 mg/ml, C) were automatically added. The luminescence originating from the seedlings was followed in time and Ca 2+ concentrations were calculated according to [START_REF] Rentel | Oxidative stress-induced calcium signaling in Arabidopsis[END_REF]. The grey marking delineates the spike in luminescence caused by the injection of the MAMPs. Shown are average Δ[Ca 2+ ] values (± standard deviation) after correction for basal Ca 2+ levels before MAMP addition. Curves shown are based on representative measurements of 4 to 6 seedlings per treatment. Each experiment was at least performed three times with similar results. A, B, E andF), elf18 (1 µM, C) or chitin (0,1 mg/ml, D) were automatically added. The luminescence originating from the seedlings was followed in time and Ca 2+ concentrations were calculated according to [START_REF] Rentel | Oxidative stress-induced calcium signaling in Arabidopsis[END_REF]. The grey marking delineates the spike in luminescence caused by the injection of the MAMPs. Shown are average Δ[Ca 2+ ] values (± standard deviation) after correction for basal Ca 2+ levels before MAMP addition (B-F) or absolute [Ca 2+ ] (A). Curves shown are based on representative measurements of 4 to 6 seedlings per treatment. Each experiment was at least performed three times with similar results.

Figure 3 Modulation of cNTP signalling does not influence the flg22-, elf18-or chitin-induced Δ[Ca 2+

]. 10 to 12-day-old aequorin-expressing Arabidopsis seedlings were pretreated for one hour with 1 mM alloxan (A), 200 μM dideoxyadenosine (B), The measurements were started and after two minutes, flg22 (1 μM) was automatically added. The luminescence originating from the seedlings was followed in time and Ca 2+ concentrations were calculated according to [START_REF] Rentel | Oxidative stress-induced calcium signaling in Arabidopsis[END_REF]. The grey marking delineates the spike in luminescence caused by the injection of the MAMPs. Shown are average Δ[Ca 2+ ] values (± standard deviation) after correction for basal Ca 2+ levels before MAMP addition. Curves shown are based on representative measurements of 4 to 6 seedlings per treatment. Each experiment was at least performed three times with similar results.

Figure 4 Modulation of iGluR-like channel function reduces the flg22-, elf18-or chitin-induced Δ[Ca 2+

]. 10 to 12-day-old aequorin-expressing Arabidopsis seedlings were pretreated for one hour with 1 mM AP-5 (A and B), 1 mM kynurenic acid (C), 1 mM L-glutamate (D and E), different amino acids (F) or a range of L-glutamate concentrations (G). The measurements were started and after two minutes, flg22 (1 μM) was automatically added. The luminescence originating from the seedlings was followed in time and Ca 2+ concentrations were calculated according to [START_REF] Rentel | Oxidative stress-induced calcium signaling in Arabidopsis[END_REF] Time point 0 minutes was harvested before MAMP addition. 1 mM L-glutamate (A) or 0,1 mg/ml chitin (B and C) were added and after 5 minutes and 15 minutes samples were taken and analysed for MAPK activation as previously described [START_REF] Saijo | Receptor quality control in the endoplasmic reticulum for plant innate immunity[END_REF]. Per sample 10-12 seedlings were used. Arrows mark the activated MAPKs on the blots. Experiments were repeated two or three times with similar results.

Figure 6 iGluR signalling and cNTP signalling act additively in elf18-induced Ca 2+ influx. 10-12-day-old aequorin expressing Arabidopsis seedlings (A) and Col-0 wild type seedlings (B) were pretreated for one hour with 1 mM L-glutamate, 1 mM alloxan or a combination of both. Aequorin based Ca 2+ measurements (A): Two minutes after initiation of luminescence detection, elf18 (1 μM) was automatically added. The luminescence originating from the seedlings was followed in time and Ca 2+ concentrations were calculated according to [START_REF] Rentel | Oxidative stress-induced calcium signaling in Arabidopsis[END_REF]. The grey marking, delineates the spike in luminescence caused by the injection of the MAMPs. Shown are the averages of the mean Δ[Ca 2+ ] of >3 biological replicates per treatment after correction for base Ca 2+ levels with the standard error of mean. MAPK assays (B): Time point 0 minutes was harvested before MAMP addition; then, 1 μM elf18 was added and after 5 minutes and 15 minutes samples were taken and analysed for MAPK activation as previously described [START_REF] Saijo | Receptor quality control in the endoplasmic reticulum for plant innate immunity[END_REF]. Per sample 10-12 seedlings were used. Arrows mark the activated MAPKs on the blots. Experiments were repeated two or three times with similar results. 10-12-day-old Arabidopsis Col-0 wild type seedlings were pretreated for one hour with 1 mM L-glutamate or 1 mM kynurenic acid. Time point 0 minutes was harvested before MAMP addition and flg22 (1 μM), elf18 (1 μM) or chitin (0,1 mg/ml) were added. Samples were taken at 30 and 60 minutes after MAMP addition and prepared for quantitative RT-PCR. PCRs were performed with primers specific for FRK1 and PHI-1 [29] and the reference gene At4G26410, The data were normalized to the reference gene and the value at time point 0 minutes of the non-pretreated samples was set to 1. The mean relative transcript accumulation of FRK1 (A) and PHI-1 (B) are presented in the graphs. Error bars represent the standard deviation of three technical replicates. The experiment was repeated three times yielding similar results. The scheme depicts a potential scenario based on our experimental results. CaM-Ca 2+ , activated (Ca 2+bound) calmodulin. For further explanation see main text. A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

= = = = = = = = = LaCl 3 = 0 = = 0 = = 0 = Blocks Ca 2+ influx channels EGTA ↑ 0 = ↑ 0 = ↑ 0 = Chelates extracellular Ca 2+ U73122 ↑ = = ↑ = = ↑ = = Inhibits phospholipase C Neomycin ↑ ↓ = ↑ ↓ = ↑ = = Inhibits phospholipase C Ruthenium Red ↑ ↓ = ↑ ↓ = ↑ ↓↓ =

Biochemical

  Journal Immediate Publication. Published on 18 Aug 2011 as manuscript BJ20111112 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20111112 A c c e p t e d M a n u s c r i p t growth [21] and hypocotyl elongation [22]. Recently, Ca 2+ influx and NO production triggered by the proteinaceous oomycete MAMP cryptogein were also found to involve iGluR-like channels [23].

  Biochemical Journal Immediate Publication. Published on 18 Aug 2011 as manuscript BJ20111112 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20111112A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 2

 2 Figure 2 No apparent contribution from internal Ca 2+ stores and VGCCs to the flg22-, elf18-and chitin-induced Δ[Ca 2+ ]. 10 to 12-day-old aequorin-expressing Arabidopsis seedlings were pretreated for one hour with 10 μM U73122 (A and B), 200 μM Ruthenium Red (C and D), 100 µM tetracaine (E) or 100 µM nifedipine (F). The measurements were started and after two minutes, flg22 (1 µM,A, B, E and F), elf18 (1 µM, C) or chitin (0,1 mg/ml, D) were automatically added. The luminescence originating from the seedlings was followed in time and Ca 2+ concentrations were calculated according to[START_REF] Rentel | Oxidative stress-induced calcium signaling in Arabidopsis[END_REF]. The grey marking delineates the spike in luminescence caused by the injection of the MAMPs. Shown are average Δ[Ca 2+ ] values (± standard deviation) after correction for basal Ca 2+ levels before MAMP addition (B-F) or absolute [Ca 2+ ] (A). Curves shown are based on representative measurements of 4 to 6 seedlings per treatment. Each experiment was at least performed three times with similar results.

  Figure 4 Modulation of iGluR-like channel function reduces the flg22-, elf18-or chitin-induced Δ[Ca 2+]. 10 to 12-day-old aequorin-expressing Arabidopsis seedlings were pretreated for one hour with 1 mM AP-5 (A and B), 1 mM kynurenic acid (C), 1 mM L-glutamate (D and E), different amino acids (F) or a range of L-glutamate concentrations (G). The measurements were started and after two minutes, flg22 (1 μM) was automatically added. The luminescence originating from the seedlings was followed in time and Ca 2+ concentrations were calculated according to[START_REF] Rentel | Oxidative stress-induced calcium signaling in Arabidopsis[END_REF]. Shown are average Δ[Ca 2+ ] values (± standard deviation) after correction for basal Ca 2+ levels before MAMP addition (B, C and E) or absolute [Ca 2+ ] (A, D). The histogram (F) shows the steady state [Ca 2+ ] or the Δ[Ca 2+ ] (nM) after flg22 (1 μM), elf18 (1 μM) or chitin (0.1 mg/ml) stimulus at the maximum of the Ca2+ transient after pretreatment for 1 hour with 1mM L-alanine [A], L-cysteine [C], L-aspartate [D], L-glutamate [E], glycine [G], L-histidine [H], L-lysine [K], L-leucine [L], L-glutamine [Q], L-tryptophan [W] and the mock control [-]. Values represent averages of 3 to 6 biological replicates with 6 individual seedlings per replicate. Statistical significant differences were assessed using a Student's t-test with treatments compared to the mock control (P<0.05) (G). For the L-glutamate concentration range (G), the average basal Ca 2+ concentration was determined using the average Ca 2+ concentration of the time points before injecting flg22. The ∆Ca 2+ value was calculated using background-corrected Ca 2+ concentrations and the values between 3.5 and 5 minutes were averaged. Each experiment was at least performed three times with similar results. Curves shown are based on representative measurements of

Figure 5

 5 Figure 5 Modulation of iGluR-like channel function or inhibition of Ca 2+ influx from the apoplast affects MAPK activation. 10-12-day-old Arabidopsis Col-0 seedlings were pretreated for one hour with 1 mM L-glutamate (A-C), 1 mM kynurenic acid (A and B), 1 mM CNQX (A and B), 1 mM LaCl 3 (C) or 10 mM EGTA (C).Time point 0 minutes was harvested before MAMP addition. 1 mM L-glutamate (A) or 0,1 mg/ml chitin (B and C) were added and after 5 minutes and 15 minutes samples were taken and analysed for MAPK activation as previously described[START_REF] Saijo | Receptor quality control in the endoplasmic reticulum for plant innate immunity[END_REF]. Per sample 10-12 seedlings were used. Arrows mark the activated MAPKs on the blots. Experiments were repeated two or three times with similar results.

Figure 7

 7 Figure 7 Modulation of iGluR-mediated Ca 2+ influx inhibits MAPK-and CDPK-dependent defense gene transcript accumulation.10-12-day-old Arabidopsis Col-0 wild type seedlings were pretreated for one hour with 1 mM L-glutamate or 1 mM kynurenic acid. Time point 0 minutes was harvested before MAMP addition and flg22 (1 μM), elf18 (1 μM) or chitin (0,1 mg/ml) were added. Samples were taken at 30 and 60 minutes after MAMP addition and prepared for quantitative RT-PCR. PCRs were performed with primers specific for FRK1 and PHI-1 [29] and the reference gene At4G26410, The data were normalized to the reference gene and the value at time point 0 minutes of the non-pretreated samples was set to 1. The mean relative transcript accumulation of FRK1 (A) and PHI-1 (B) are presented in the graphs. Error bars represent the standard deviation of three technical replicates. The experiment was repeated three times yielding similar results.

Figure 8 A

 8 Figure 8 A model for MAMP-triggered iGluR-mediated Ca 2+ influx in Arabidopsis thaliana.The scheme depicts a potential scenario based on our experimental results. CaM-Ca 2+ , activated (Ca 2+bound) calmodulin. For further explanation see main text.

G

  Figure 4 A

  Publication. Published on 18 Aug 2011 as manuscript BJ20111112 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20111112 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

  Publication. Published on 18 Aug 2011 as manuscript BJ20111112 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20111112

  Figure 7

Table 1 Overview of alterations in intracellular Ca 2+ levels upon MAMP treatment in the presence of compounds modulating Ca 2+ transport a . flg22 elf18 Chitin Pre-treatment Base Peak Rec Base Peak Rec Base Peak Rec Action -

 1

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2011 The Authors Journal compilation © 2011 Portland Press Limited

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited iGluRs mediate MAMP-triggered calcium influx in Arabidopsis

Acknowledgements

We thank Justine Lorek for providing protocols and primer sequences for qRT-PCR and MAPK assays. We are grateful to the lab of Tina Romeis for performing CDPK activity assays. We acknowledge Dierk Scheel and Mark Knight for kindly providing seeds of the 35S-aequorin line. We apologize to authors whose work could not be cited due to space constraints.

Funding

Work in the lab of RP is supported by grants from the Max-Planck society and the Deutsche Forschungsgemeinschaft (DFG; SFB670).


J Jasper Deuring
email: j.deuring@erasmusmc.nl

Colin De Haar

Chantal L Koelewijn

Ernst J Kuipers

Maikel P Peppelenbosch

C Janneke Van Der Woude

Absence of ABCG2-mediated mucosal detoxification in patients with active inflammatory bowel disease is due to impeded protein folding

Keywords: IBD, inflammatory bowel disease, CD, Crohn's disease, UC, ulcerative colitis, IEC, intestinal epithelium cells, NO, nitric oxide, ER, endoplasmic reticulum, PDI, protein-disulfide isomerase, GFP, green fluorescent protein, RFP, red fluorescent protein, 293t-AB, HEK293t cells transiently transfected with GFP-ABCG2, FFPE, formalin fixed paraffin embedded, IHC, immunohistochemistry

Introduction

The intestinal epithelium is a single-cell physical barrier between the gut luminal content and mucosa, important for detection [START_REF] Abreu | Toll-like receptor signalling in the intestinal epithelium: how bacterial recognition shapes intestinal function[END_REF] and defence [START_REF] Perminow | Defective paneth cell-mediated host defense in pediatric ileal Crohn's disease[END_REF][START_REF] Salzman | Paneth cells, defensins, and the commensal microbiota: a hypothesis on intimate interplay at the intestinal mucosa[END_REF] against microbes. Intestinal epithelial cells (IEC) are constantly exposed to a large variety xenobiotic substances, including microbes and their products as well as exogenous (e.g. drugs or food) and endogenous (e.g. bile acids) toxic substances present in the luminal content. IEC utilize various mechanisms to maintain epithelial integrity in face of the xenobiotic challenge. They are protected against exogenous and endogenous toxic compounds by, among others, proteins from the ATP-binding cassette (ABC) transporter family. This family includes the P-glycoprotein/Multidrug Resistance (P-gp/MDR1/ABCB1), Multidrug Resistance-associated Protein-1 (MRP1/ABCC1), Multidrug Resistance-associated Protein-2 (MRP2/ABCC2) and Breast Cancer Resistance Protein (BCRP/ABCG2) [START_REF] Dietrich | ABC of oral bioavailability: transporters as gatekeepers in the gut[END_REF][START_REF] Leslie | Multidrug resistance proteins: role of P-glycoprotein, MRP1, MRP2, and BCRP (ABCG2) in tissue defense[END_REF]. Especially the homodimeric xenobiotic transporter ABCG2, which specifically localises to the IEC apical membrane, is widely considered to be especially important in combating luminal-derived xenobiotic stress [START_REF] Robey | ABCG2: a perspective[END_REF] [START_REF] Englund | Efflux transporters in ulcerative colitis: decreased expression of BCRP (ABCG2) and Pgp (ABCB1)[END_REF]. Disruption of IEC integrity leads to inflammatory lesions, which are a hallmark of active inflammation in patients with inflammatory bowel disease (IBD). Appropriate response to such disruption requires rapid mucosal healing by cell division, differentiation and maturation to restore barrier function [START_REF] Dignass | Mechanisms and modulation of intestinal epithelial repair[END_REF][START_REF] Podolsky | Healing the epithelium: solving the problem from two sides[END_REF]. Active inflammation in patients with IBD has been associated with dramatically reduced expression of ABC-transporters in IEC and the resulting diminished capacity to deal with luminal xenobiotic challenge is thought to interfere with the efficiency of regenerative responses [START_REF] Englund | Efflux transporters in ulcerative colitis: decreased expression of BCRP (ABCG2) and Pgp (ABCB1)[END_REF][START_REF] Petrovic | Effect of endotoxin on the expression of placental drug transporters and glyburide disposition in pregnant rats[END_REF][START_REF] Poller | Regulation of BCRP (ABCG2) and P-glycoprotein (ABCB1) by cytokines in a model of the human blood-brain barrier[END_REF][12]. Further compounding this problem is that several medications used in IBD are specific substrates of ABCG2. Hence understanding of the mechanisms governing ABCG2 expression is highly relevant for devising improved therapy [START_REF] Krishnamurthy | Role of ABCG2/BCRP in biology and medicine[END_REF][START_REF] Doyle | A multidrug resistance transporter from human MCF-7 breast cancer cells[END_REF]. Nevertheless, the molecular mechanisms driving ABCG2 expression during inflammatory responses remain obscure at best. Generally, excreted and membrane-bound proteins are translated in the endoplasmic reticulum (ER). If ER function is disrupted, proteins can accumulate inside the ER and this can initiate a specific ER stress syndrome denoted the unfolded protein response. Recently, abnormalities in the ER stress response pathways have been associated with the pathogenesis of IBD [START_REF] Heazlewood | Aberrant mucin assembly in mice causes endoplasmic reticulum stress and spontaneous inflammation resembling ulcerative colitis[END_REF][START_REF] Kaser | XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease[END_REF][START_REF] Shkoda | Interleukin-10 blocked endoplasmic reticulum stress in intestinal epithelial cells: impact on chronic inflammation[END_REF]. Proper function of the ABCG2 protein depends on Nglycosylation and the formation of inter/intra molecular disulfide bonds [START_REF] Nakagawa | Disruption of N-linked glycosylation enhances ubiquitin-mediated proteasomal degradation of the human ATP-binding cassette transporter ABCG2[END_REF][START_REF] Wakabayashi | Intramolecular disulfide bond is a critical check point determining degradative fates of ATP-binding cassette (ABC) transporter ABCG2 protein[END_REF]. These bonds are formed inside the ER by protein-disulfide isomerases (PDI). Inflammatory conditions, such as increased nitric oxide production, can dramatically reduce the efficiency of these PDI, which can initiate misfolding of various proteins leading to ER stress [START_REF] Oyadomari | Nitric oxide-induced apoptosis in pancreatic beta cells is mediated by the endoplasmic reticulum stress pathway[END_REF][START_REF] Uehara | S-nitrosylated protein-disulphide isomerase links protein misfolding to neurodegeneration[END_REF]. As correct ABCG2-protein folding is necessary for its functionality, we hypothesized that mucosal inflammation in the setting of IBD alters proper ABCG2-protein folding and thereby reduces its apical expression. In this study we describe a novel mechanism by which ABCG2 protein expression is decreased during active inflammation.

Experimental procedures

Patient materials

Formalin-fixed paraffin-embedded (FFPE) colonic biopsies were collected from nine healthy individuals and from 76 patients: 25 with Crohn's disease (CD), 36 with ulcerative colitis (UC), 10 with ischemic colitis and 5 with infectious colitis. Biopsies throughout the small intestine were collected from 17 other healthy individuals and 34 CD patients. For mRNA isolation freshly-frozen biopsies were collected from six healthy individuals and from 15 patients: 8 with inactive IBD and 7 with active IBD. The demographic characteristics, number of biopsies per group, duration of disease and medication use are described in supporting Tables 1, 2 and 3 (included in the supporting section).

Immunohistochemistry and scoring

The FFPE sections were immunohistologically stained using the primary antibodies BXP-21 anti-ABCG2 (Santa Cruz Biotechnology, Santa Cruz, CA) and BiP anti-GRP78 (Cell Signalling, Boston, USA) according to the manufacturers' protocol (included in the supporting section). The expression of ABCG2 and GRP78 in IEC was scored by intensity over the whole slide by two independent observers, and discrepancies were re-assessed to come to a final agreement. Individual scores were used for statistical analyses, whereas they were combined into three subgroups for graphic representation. For ABCG2 positive staining at the apical membrane of the epithelial cells was defined as positive if >50% of the IEC were positive, mild 15-50%, or negative <15%. Immunoreactivity for GRP78 was also defined in terms of positive IEC, but now the percentage were >70% defined as positive, 20-70% mild, <20% negative. mRNA isolation and quantitative PCR mRNA was isolated from colonic biopsies, which were taken from CD and UC patients, using the NucleoSpin Extract II mRNA isolation kit (Macherey Nagel, Düren, Germany). Gene expression of GapdH, IL8, IL1beta, ABCG2, villin, and ER stress associated gene CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) [START_REF] Oyadomari | Nitric oxide-induced apoptosis in pancreatic beta cells is mediated by the endoplasmic reticulum stress pathway[END_REF] were measured using quantitative PCR (qPCR, IQ5, Bio-Rad). Villin and ABCG2 are both IEC specific, thus ABCG2 mRNA is corrected for Villin to prevent underestimation in biopsies with less IEC. A detailed protocol and the primer sequences involved are presented in the supporting section.

Cell culture

HEK293t (ATCC# CRL-1573) cells were cultured according to routine procedures. Transient transfection was performed using Polyethylenimine (PEI, Polysciences, Eppelheim, Germany). S-nitroso-N-acetylpenicillamine (SNAP, Sigma-Aldrich) was used as a NO donor. Tunicamycin from Streptomyces sp. (Sigma-Aldrich) was used as a positive control for ER stress induction. The cell culture supernatant nitrate concentrations were measured using Griess Reagents (Cayman Chemical, Michigan, USA) according to the manufacturers protocol. Additional cell culture methods are described in the supporting section.

XBP1 splicing ER stress and unfolded protein response activity was assessed by identify mature XBP1 mRNA and its spliced variant [START_REF] Kaser | XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease[END_REF] (included in the supporting section).

Construct cloning

To investigate ABCG2 functionality we created a green fluorescent protein (GFP)-ABCG2 fusion construct (included in the supporting section).

Fluorescent microscopy

The GFP-tagged proteins were examined using a confocal microscope (LSM510META, Zeiss). For the transport assay, nuclear Hoechst 33342 (4 µM, Sigma-Aldrich) accumulation was determined at 30 s intervals during a total of 12 min. The ER was stained with ER-RFP (Red Fluorescent Protein) using ER-Tracker (Molecular Probes, Paisley, UK) according to the manufacturer's instruction. We analyzed the GFP tagged proteins with a 488 nm laser, Hoechst with a 405 nm laser and the ER-RFP with 543 nm laser. A specific ABCG2 transport inhibitor Ko143 (2 µM, Sigma-Aldrich) was used to indentify ABCG2 transport specificity.

SDS-page and fluorescent immunoblotting

For checking correct length of fusion proteins, whole cell lysates were analysed on immunoblot from standard SDS-page gel, using antibodies against ABCG2 (BXP-21, Santa Cruz Biotechnology) and GFP (polyclonal, rabbit IgG, Invitrogen) (included in the supporting section).

Statistics and software

Statistical analyses were performed using the SPSS 11.0 software package for Windows. Data on different patient groups were compared using the Mann-Witney U test with Kruskal-Wallis post test. Using univariate and multivariate multinomial logistic regression analysis, we examined the association between colitis and GRP78 and ABCG2 expression, adjusting for age and duration of disease. A two-tailed p value <0.05 was accepted as statistically significant. Images were composed using Adobe Photoshop CS3.

Results

Colonic inflammation reduces apical ABCG2 expression in IEC

To investigate ABCG2 expression during human intestinal inflammation, ABCG2 expression in colonic biopsies was qualitatively assessed employing immunohistochemistry (IHC). As expected IEC in biopsies of control subjects as well as of IBD patients in remission (with no active inflammation) show strong specific staining for ABCG2 at the apical surface (Figure 1A). In contrast, apical IEC ABCG2 staining is dramatically reduced in patients with active IBD, in the colon as well as in the small intestine, and also in biopsies from ischemic colitis and infectious colitis patients (Figure 1A). Thus diverse colonic inflammatory responses are uniformly characterised by down regulation of apical ABCG2 levels, suggesting that processes common to these alternative modes of inflammation underlie the absence of apical ABCG2 intestinal inflammation. In apparent agreement, quantitative study of ABCG2 levels in the human colon reveals significantly less ABCG2 expression in active colitis (24% positive expression, 13/54), ischemic colitis (10% positive staining, 1/10) and infectious colitis (0% positive staining 0/14) as compared to either inactive colitis (80% positive expression, 53/67) or control tissues (100% positive expression, 9/9), all p<0.001 (see Figure 1B). There is no significant difference in ABCG2 levels between active CD (27%, 6/22) vs. active UC (22%, 7/32) and inactive CD (67%, 18/27) vs. inactive UC (88%, 35/40). As a control, we examined left-and right-sided biopsies from IBD patients and controls to investigate a confounding possible differential expression of ABCG2 based on position in the colon, but this analysis demonstrates that only the degree of inflammation is an important factor guiding apical ABCG2 levels. Thus inflammation per se is associated with a marked reduction of apical ABCG2 expression in the human colon.

Small bowel inflammation is associated with down regulation of apical ABCG2 expression

Mirroring the findings obtained in the human colon, small bowel biopsy specimens obtained from CD patients with active disease by double balloon endoscopy exhibit markedly less ABCG2 staining when compared to biopsies obtained from patients with inactive disease or no intestinal inflammatory phenomena (see Figure 1A). In active CD, there is significantly less small bowel ABCG2 staining (75% negative staining, 19/25) as compared to inactive CD (11% negative staining, 1/9) and controls (8% negative staining, 2/25), both P<0.05 (see Figure 1C). We find no significant differences between inactive CD and controls. In all cases current drug use did not influence the expression of ABCG2. We conclude that human intestinal inflammation is accompanied by reduced expression of xenobiotic transporter ABCG2 and experiments were initiated to identify the underlying molecular mechanisms.

Intestinal inflammation acts to reduce ABCG2 via a post-transcriptional mechanism

To delineate the molecular events reducing the capacity of the intestine to cope with xenobiotic challenge we first determined whether genomic or post-transcriptional processes are involved in this effect. To this end, we measured ABCG2 mRNA levels in freshly frozen colon biopsies from healthy individuals, inactive and active IBD patients. Importantly, ABCG2 mRNA, levels in IEC, do not differ between these three groups (all p>0.8). The expression, however, of the pro-inflammatory cytokine IL8 and IL1beta are 10and 8-fold higher (p<0.05) in biopsies with active mucosal inflammation (Figure 2) and thus technical issues do not underlie the absence of intestinal inflammation-induced effects on apical ABCG2 expression. Hence we are forced to conclude that down modulation of ABCG2 in human intestinal inflammation is brought about by a posttranscriptional process.

Activation of the protein unfolded protein response accompanies human intestinal inflammation

As it is becoming increasingly clear that activation of the unfolded protein response accompanies inflammatory processes and that ER stress may interfere with proper production of complexly folded proteins (the "half" transporter ABCG2 transverses the membrane 6 times), we examined whether the ER stress pathway is activated in intestinal inflammation and to which extent this activation mirrors the down regulation of ABCG2. To this end we employ the expression of the bona fide ER stress marker GRP78. Representative images of the GRP78 immunohistochemical analyses of the same biopsies as stained for ABCG2 are shown in Figure 3A. There is a significant increase in GRP78 expression in active colitis (32% positive expression, 16/54), ischemic colitis (90% positive staining, 9/10) and infectious colitis (79% positive staining 11/14), compared to inactive colitis (0% positive expression, 0/67) and control tissues (0% positive expression, 0/9), all p<0.001 (see Figure 3B). There are no significant differences in GRP78 expression between active CD and active UC or between inactive CD and inactive UC. Also there are no differences in GRP78 expression in left-and right-sided colon biopsies taken from the same patient. We conclude that colonic inflammation is accompanied by ER stress response which is absent in the un-inflamed colon. In the small bowel biopsies of active CD patients there is increased GRP78 expression (29% positive expression, 6/21) compared to inactive CD patients (0% positive staining, 0/9) and controls (8% positive expression, 2/25) p<0.05 (see Figures 3A and3C). In all actively inflamed tissue samples elevated GRP78 expression is evident, irrespective of disease type or location. ER stress during intestinal inflammation is further confirmed measuring the mRNA expression of ER stress associated gene, CHOP, in colonic biopsies. In biopsies taken from actively inflamed mucosa a significant increase in CHOP mRNA expression was measured, p<0.01 (see Figure 2B). Moreover, an increased ratio of spliced XBP1 mRNA was detected in patients with active intestinal inflammation (Figure 2C). We conclude that activation of the unfolded protein response and intestinal inflammation are highly associated processes and we initiated experiments to investigate whether ER stress explains down regulation of ABCG2 in intestinal inflammation.

Impaired ABCG2 function in ER stressed cells

To investigate the effect of activated ER stress on ABCG2 protein expression and function, we created a GFP-ABCG2 fusion protein. To this end, ABCG2 cDNA was cloned into a pEGFP-C1 vector which led to the production of GFP-ABCG2 after transfection (Figure 4A). The NO-donor SNAP was used to ascertain whether NO was able to induce ER stress in GFP-ABCG2 transfected HEK293t (293t-AB) cells. The Griess assay reveals SNAP application induces robust increases in NO 2 concentration, a good proxy measure for NO production (see Figure 4B). The validity of assay is demonstrated by experiments showing an increase in the ratio of unspliced XBP1 mRNA over spliced XBP1 mRNA nine h after application of 0.625 mM SNAP (see Figure 4C). In agreement administration of tunicamycin, a well established ER stress response inducer, increases the XBP1 splicing ratio to approximately the same level as SNAP application (supporting Figure 1B). Consistent with high ABCG2 transport activity, administration of Hoechst 33342 does not stain the nuclei of the 293t-AB cells (Figure 5A), also demonstrating the functionality of the ABCG2-GFP fusion protein in xenobiotic transport. Nevertheless, 16 h after addition of the ER-stress inducers SNAP or tunicamycin, the nuclei of 293t-AB cells become Hoechst 33342 positive (Figure 5A and supporting movies 1, 2 and 3). As shown in Figure 5B Hoechst 33342 also stains the nucleus blue in 293t-AB transfected cells in the presence of a specific ABCG2 inhibitor Ko143 (see also supporting movie 4). No altered GFP-ABCG2 protein function is found when SNAP or tunicamycin is added 48 h after transfection (see 1C). Hence, in this over expressing model system ER stress provokes a very strong decrease in ABCG2 function, in agreement with a scheme in which inflammation-dependent induction of ER stress is causatively related to a reduced capacity to deal with xenobiotic challenge.

In support for such a model, all images from 293t-AB cells with ER stress reveal reduced apical membrane expression of GFP-ABCG2, with remaining intracellular expression. In order to ascertain whether GFP-ABCG2 remains in the ER after stress induction, we stained the ER with ER-RFP. When ER stress was present some GFP-ABCG2 colocalized with ER-RFP, whereas the majority was present in other cellular compartments (Figure 5C). As shown in supporting Figure 2 the localization and expression of non-ER dependent GFP-tagged proteins (GFP-betaCatenin, GFP-H2B) was not changed during ER stressed conditions. Thus protein folding difficulties seems incompatible with functional expression of ABCG2.

Discussion

The data presented in the current study are a consistent with a model in which intestinal inflammation drives an epithelial misfolded protein response, which in turn interferes with the ABCG2 expression and function leading to a diminished capacity of the epithelial compartment to cope with xenobiotic stress. Colonic and small bowel biopsies from IBD patients with active disease and those from patients with ischemic and infectious colitis all show reduced apical ABCG2 expression in IEC compared to controls and IBD patients in remission. The decline of ABCG2 expression in the presence of active inflammation in IBD patients corresponds with the findings of two studies done exclusively on UC patients [START_REF] Englund | Efflux transporters in ulcerative colitis: decreased expression of BCRP (ABCG2) and Pgp (ABCB1)[END_REF][START_REF] Gutmann | Breast Cancer Resistance Protein and P-Glycoprotein Expression in Patients with Newly Diagnosed and Therapy-Refractory Ulcerative Colitis Compared with Healthy Controls[END_REF]. In addition, we found that ABCG2 expression is not affected by medication use, patient age, or location of the inflammation. Expression of ABCG2, like that of many other proteins, can be regulated at various levels. In addition to a decline in ABCG2 protein expression, others have described a reduction in ABCG2 mRNA expression in actively inflamed intestine [START_REF] Englund | Efflux transporters in ulcerative colitis: decreased expression of BCRP (ABCG2) and Pgp (ABCB1)[END_REF][START_REF] Gutmann | Breast Cancer Resistance Protein and P-Glycoprotein Expression in Patients with Newly Diagnosed and Therapy-Refractory Ulcerative Colitis Compared with Healthy Controls[END_REF]. We corrected the ABCG2 mRNA expression for Villin to prevent underestimation of ABCG2 expression, since both genes are specific for IEC. Our results show no reduction of ABCG2 mRNA expression in biopsies with active mucosal inflammation. However, since the sample size used here is smaller than those in previous studies, we cannot completely rule out the contribution of reduced mRNA expression to the decline in ABCG2 protein expression. As such our findings merely imply that the decline in mRNA expression probably does not solely account for the complete loss of ABCG2 protein expression discernable on our IHC images and that post-transcriptional mechanisms must be involved. The observation that unfolded protein responses are both a general and specific to intestinal inflammation suggest a causal link between ER stress and diminished apical ABCG2 levels. Indeed, ABCG2 is a "half" transporter, 6 times membrane spanning protein, functionally critically dependent on homodimerisation, N-linked glycosylation and various disulfide bonds and is thus is a likely early victim of ER stress responses [START_REF] Nakagawa | Disruption of N-linked glycosylation enhances ubiquitin-mediated proteasomal degradation of the human ATP-binding cassette transporter ABCG2[END_REF][START_REF] Wakabayashi | Intramolecular disulfide bond is a critical check point determining degradative fates of ATP-binding cassette (ABC) transporter ABCG2 protein[END_REF]. Disrupted protein-folding mechanisms that can lead to ER stress during active inflammation have recently been linked to IBD [START_REF] Kaser | XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease[END_REF][START_REF] Kolios | Nitric oxide in inflammatory bowel disease: a universal messenger in an unsolved puzzle[END_REF][START_REF] Mcguckin | ER Stress and the Unfolded Protein Response in Intestinal Inflammation[END_REF] and may well involve reduced capacity of the IEC compartment to deal with the xenobiotic stress and thus in turn interfere with demanding regenerative responses that an adequate response to IBD-related intestinal challenges requires. Our inflammation links to unfolded protein responses remains unclear, but associated with inflammatory responses is an excessive production of NO, which in turn is predicted to disturb the function of redox-state sensitive disulfide-bonding chaperones like PDI that are necessary for proper protein folding [START_REF] Townsend | Nitrosative stress-induced s-glutathionylation of protein disulfide isomerase leads to activation of the unfolded protein response[END_REF]. In all biopsies with active inflammation (i.e. IBD and non-IBD related), the GRP78 expression is significantly higher than in the biopsies of IBD patients in remission, and in those of healthy controls. Furthermore, expression of CHOP mRNA (an ER stress marker) is increased in biopsies with active mucosal inflammation. In addition to this, biopsies with active inflammation reveal an increased ration of spliced XBP1 mRNA expression. Thus factors common to multiple types of inflammation must be involved here and as NO production is one of the few cellular responses described to generally accompany inflammation, it is tempting to attribute a important role to excessive NO generation in inflammation-related induction of ER stress responses [START_REF] Kolios | Nitric oxide in inflammatory bowel disease: a universal messenger in an unsolved puzzle[END_REF][START_REF] Townsend | Nitrosative stress-induced s-glutathionylation of protein disulfide isomerase leads to activation of the unfolded protein response[END_REF][START_REF] Gotoh | Nitric oxide and endoplasmic reticulum stress[END_REF][START_REF] Herulf | Increased luminal nitric oxide in inflammatory bowel disease as shown with a novel minimally invasive method[END_REF]. Indeed, we observe elevated XBP1 splicing [START_REF] Kaser | XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease[END_REF] following application of the NO-donor, suggesting that NO can directly affect protein-folding mechanisms [START_REF] Oyadomari | Nitric oxide-induced apoptosis in pancreatic beta cells is mediated by the endoplasmic reticulum stress pathway[END_REF] and induce ER stress. In the current study we show that the reduction in ABCG2 expression is inflammation-associated, which fits with the observations that ABCG2 expression is reduced in IBD-unrelated inflammatory conditions in other organs [START_REF] Petrovic | Effect of endotoxin on the expression of placental drug transporters and glyburide disposition in pregnant rats[END_REF]12,[START_REF] Petrovic | Regulation of drug transporters during infection and inflammation[END_REF]. Other IBD associated proteins are also reduced in protein expression during active intestinal inflammation [29][30][START_REF] Thibault | Down-regulation of the monocarboxylate transporter 1 is involved in butyrate deficiency during intestinal inflammation[END_REF]. Further research is needed to investigate the role impeded protein folding and the effect of this on cellular processes. Especially, ABCG2mediated efflux of toxic components is thought to protect the epithelium of the gut and the underlying tissue to xenobiotic challenge. Importantly, as xenobiotic stress is directly linked to genotoxicity our results may also help explaining the still poorly understood relation between intestinal inflammation and colorectal cancer [START_REF] Itzkowitz | Inflammation and cancer IV. Colorectal cancer in inflammatory bowel disease: the role of inflammation[END_REF]. Decreased ABCG2 expression may have other deleterious consequences as well. Sulfasalazine, a derivate of mesalazine (5-ASA) that is commonly used in the treatment of IBD patients, is actively excluded from the epithelial compartment by ABCG2 [33]. Rodents with reduced IEC ABCG2 expression have increased 5-ASA trough levels [START_REF] Zaher | Breast cancer resistance protein (Bcrp/abcg2) is a major determinant of sulfasalazine absorption and elimination in the mouse[END_REF]. In addition to its antiinflammatory effect, accumulation of sulfasalazine can be toxic for epithelial cells [35][START_REF] Terdiman | 5-Aminosalicylic acid therapy and the risk of colorectal cancer among patients with inflammatory bowel disease[END_REF]. Thus reduced expression and function of ABCG2 may therefore greatly enhance the sensitivity of IEC to sulfasalazine and limit the use of this compound for the treatment of IBD. Hence understanding the mechanisms governing ABCG2 expression are highly relevant for efforts with respect to rational design of novel therapy. In conclusion, IEC ABCG2 protein expression is decreased in the inflamed bowel probably as a result of incorrect protein folding due to inflammation-driven ER dysfunction. Thus a novel paradigm explaining xenobiotic stress during inflammation emerges, in which intestinal inflammation actives the unfolded protein response in turn abrogating defences against xenobiotic challenge by impairing ABCG2 expression and function. Colon, A) colon biopsy from an IBD patient with active inflammation. Colon, ISC) colon biopsy from a patient with ischemic colitis. Colon, IFC) colon biopsy from a patient with infectious colitis. SB Ctr) small bowel biopsy from a healthy individual. SB, IA) small bowel biopsy from a Crohn's disease patient in remission. SB, A) small bowel biopsy from a Crohn's disease patient with active inflammation. The ABCG2 expression is represented by red-brown staining on the apical membrane of IEC. Original magnification is 400x. B) Decrease in the IEC fraction of colonic biopsies with ABCG2 expression during active inflammation. C) Actively inflamed small bowel displays reduced expression of ABCG2. B) and C) All intestinal biopsies were double blindly examined as negative, moderate or positive for ABCG2 membrane immune reactivity. Mann-Whitney U tests and Kruskal-Wallis tests were used for statistical analysis. Colon, A) colon biopsy from an IBD patient with active inflammation. Colon, ISC) colon biopsy from a patient with ischemic colitis. Colon, IFC) colon biopsy from a patient with infectious colitis. SB Ctr) small bowel biopsy from a healthy individual. SB, IA) small bowel biopsy from a Crohn's disease patient in remission. SB, A) small bowel biopsy from a Crohn's disease patient with active inflammation. Expression of GRP78 is detected as redbrown colour and is present in the cytoplasm of IEC and mononuclear cells in the lamina propria. Original magnification is 400x. B) Increased in the IEC fraction of colonic biopsies with elevated GRP78 expression during active inflammation. C) Actively inflamed small bowel displays elevated GRP78 expression. B) and C) The biopsies were double blindly examined as positive, moderate or negative according the amount of positive GRP78 immune reactivity. Mann-Whitney U tests and Kruskal-Wallis tests were used for statistical analysis.

5

  Biochemical Journal Immediate Publication. Published on 25 Aug 2011 as manuscript BJ20111281 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20111281 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

7

  Biochemical Journal Immediate Publication. Published on 25 Aug 2011 as manuscript BJ20111281 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20111281 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited supporting Figure

8

  Biochemical Journal Immediate Publication. Published on 25 Aug 2011 as manuscript BJ20111281 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20111281 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

Biochemical

  Journal Immediate Publication. Published on 25 Aug 2011 as manuscript BJ20111281 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20111281 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. Biochemical Journal Immediate Publication. Published on 25 Aug 2011 as manuscript BJ20111281 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20111281 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

Figure legends Figure 1 :

 legends1 Figure legends

Figure 2 :

 2 Figure 2: mRNA expression from intestinal biopsies.A) No difference in ABCG2 mRNA expression in colonic biopsies. Relative mRNA (deltadeltaCt) expression corrected for Villin. Villin and ABCG2 are both IEC specific, thus ABCG2 mRNA is corrected for Villin to prevent underestimation in biopsies with less IEC. B) Increased pro-inflammatory cytokine expression in colonic biopsies from patients with active inflammation. Relative mRNA expression (deltadeltaCt) corrected for GapdH. IL8 and IL1beta are pro-inflammatory cytokines and CHOP is an ER stress related gene. . A) and B) Control are healthy individuals (n=6), ia IBD are biopsies with inactive inflammation (n=8), a IBD are biopsies with active inflammation (n=7). Mann-Whitney U tests and Kruskal-Wallis tests were used for statistical analysis. C) XBP1 mRNA splicing. Agarose gel image of XBP1 splicing PCR products. Spliced XBP1 is an indicator for increased ER stress. A is active IBD, IA is inactive IBD and H is healthy control.

Figure 3 :

 3 Figure 3: Increased GRP78 expression in actively inflamed intestinal biopsies.A) Representative images of intestinal biopsies IHC stained for GRP78. Colon, Ctr) colon biopsy from healthy individual. Colon, IA) colon biopsy from an IBD patient in remission. Colon, A) colon biopsy from an IBD patient with active inflammation. Colon, ISC) colon biopsy from a patient with ischemic colitis. Colon, IFC) colon biopsy from a patient with infectious colitis. SB Ctr) small bowel biopsy from a healthy individual. SB, IA) small bowel biopsy from a Crohn's disease patient in remission. SB, A) small bowel biopsy from a Crohn's disease patient with active inflammation. Expression of GRP78 is detected as redbrown colour and is present in the cytoplasm of IEC and mononuclear cells in the lamina propria. Original magnification is 400x. B) Increased in the IEC fraction of colonic biopsies with elevated GRP78 expression during active inflammation. C) Actively inflamed small bowel displays elevated GRP78 expression. B) and C) The biopsies were double blindly examined as positive, moderate or negative according the amount of positive GRP78 immune reactivity. Mann-Whitney U tests and Kruskal-Wallis tests were used for statistical analysis.

Figure 4 :

 4 Figure 4: Nitric oxide induced ER stress reduces ABCG2 protein expression and function.A) Western blot analysis showing the expression of GFP-ABCG2 protein using the antibodies BXP21 (anti-ABCG2) and anti-GFP. Tubulin (Tub) and beta-actin (b-Act) were used as loading control. MM is molecular marker.

Figure 5 :

 5 Figure 5: Reduced ABCG2 activity and less membrane localization in ER stressed cells. Hoechst 33342 (4 µM) DNA binding was measured to study the activity of ACBG2. A) ABCG2 transfected cells were exposed to SNAP, tunicamycin or solvent for 16 h after which Hoechst 33343 binding was studied. See supporting Movies 1, 2 and 3. Original magnification 400x. B) The specific inhibitor of ABCG2, Ko143 (2 µM) added 12 min after Hoechst 33342 (4 µM)), was used as a positive control for ABCG2 activity. The accompanying movie is supporting Movie 4. Original magnification 400x. C) 293t-AB cells were stained with ER-RFP. ER stress was induced by addition of SNAP or tunicamycin 16 h before ER-RFP staining. All confocal images are representative of at least three independent experiments. Original magnification 400x.

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2011 The Authors Journal compilation © 2011 Portland Press Limited

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

Acknowledgements

The authors thank Dr. R. Smits for providing the GFB-betaCatenin and GFP-H2B fusion constructs. The authors also thank Dr. H. van Dekken and Dr. K. Biermann for pathological assessment of the intestinal cross sections.