De-regulated expression of the plant glutamate receptor homolog AtGLR3.1 impairs long-term Ca2+-programmed stomatal closure

Cytosolic Ca²⁺ ([Ca²⁺]_cyt) mediates diverse cellular responses in both animal and plant cells in response to various stimuli. Calcium oscillation amplitude and frequency control gene expression. In stomatal guard cells, [Ca²⁺]_cyt has been shown to regulate stomatal movements, and a defined window of Ca²⁺ oscillation kinetic parameters encodes necessary information for long‐term stomatal movements. However, it remains unknown how the encrypted information in the cytosolic Ca²⁺ signature is decoded to maintain stomatal closure. Here we report that the Arabidopsis glutamate receptor homolog AtGLR3.1 is preferentially expressed in guard cells compared to mesophyll cells. Furthermore, over‐expression of AtGLR3.1 using a viral promoter resulted in impaired external Ca²⁺‐induced stomatal closure. Cytosolic Ca²⁺ activation of S‐type anion channels, which play a central role in Ca²⁺‐reactive stomatal closure, was normal in the AtGLR3.1 over‐expressing plants. Interestingly, AtGLR3.1 over‐expression did not affect Ca²⁺‐induced Ca²⁺ oscillation kinetics, but resulted in a failure to maintain long‐term ‘Ca²⁺‐programmed’ stomatal closure when Ca²⁺ oscillations containing information for maintaining stomatal closure were imposed. By contrast, prompt short‐term Ca²⁺‐reactive closure was not affected in AtGLR3.1 over‐expressing plants. In wild‐type plants, the translational inhibitor cyclohexamide partially inhibited Ca²⁺‐programmed stomatal closure induced by experimentally imposed Ca²⁺ oscillations without affecting short‐term Ca²⁺‐reactive closure, mimicking the guard cell behavior of the AtGLR3.1 over‐expressing plants. Our results suggest that over‐expression of AtGLR3.1 impairs Ca²⁺ oscillation‐regulated stomatal movements, and that de novo protein synthesis contributes to the maintenance of long‐term Ca²⁺‐programmed stomatal closure.     

An Arabidopsis berberine bridge enzyme‐like protein specifically oxidizes cellulose oligomers and plays a role in immunity

The plant cell wall is the barrier that pathogens must overcome to cause a disease, and to this end they secrete enzymes that degrade the various cell wall components. Due to the complexity of these components, several types of oligosaccharide fragments may be released during pathogenesis and some of these can act as damage‐associated molecular patterns (DAMPs). Well‐known DAMPs are the oligogalacturonides (OGs) released upon degradation of homogalacturonan and the products of cellulose breakdown, i.e. the cellodextrins (CDs). We have previously reported that four Arabidopsis berberine bridge enzyme‐like (BBE‐like) proteins (OGOX1–4) oxidize OGs and impair their elicitor activity. We show here that another Arabidopsis BBE‐like protein, which is expressed coordinately with OGOX1 during immunity, specifically oxidizes CDs with a preference for cellotriose (CD3) and longer fragments (CD4–CD6). Oxidized CDs show a negligible elicitor activity and are less easily utilized as a carbon source by the fungus Botrytis cinerea. The enzyme, named CELLOX (cellodextrin oxidase), is encoded by the gene At4 g20860. Plants overexpressing CELLOX display an enhanced resistance to B. cinerea, probably because oxidized CDs are a less valuable carbon source. Thus, the capacity to oxidize and impair the biological activity of cell wall‐derived oligosaccharides seems to be a general trait of the family of BBE‐like proteins, which may serve to homeostatically control the level of DAMPs to prevent their hyperaccumulation.     

Ethylene production in Botrytis cinerea‐ and oligogalacturonide‐induced immunity requires calcium‐dependent protein kinases

Plant immunity against pathogens is achieved through rapid activation of defense responses that occur upon sensing of microbe‐ or damage‐associated molecular patterns, respectively referred to as MAMPs and DAMPs. Oligogalacturonides (OGs), linear fragments derived from homogalacturonan hydrolysis by pathogen‐secreted cell wall‐degrading enzymes, and flg22, a 22‐amino acid peptide derived from the bacterial flagellin, represent prototypical DAMPs and MAMPs, respectively. Both types of molecules induce protection against infections. In plants, like in animals, calcium is a second messenger that mediates responses to biotic stresses by activating calcium‐binding proteins. Here we show that simultaneous loss of calcium‐dependent protein kinases CPK5, CPK6 and CPK11 affects Arabidopsis thaliana basal as well as elicitor‐ induced resistance to the necrotroph Botrytis cinerea, by affecting pathogen‐induced ethylene production and accumulation of the ethylene biosynthetic enzymes 1‐aminocyclopropane‐1‐carboxylic acid (ACC) synthase 2 (ACS2) and 6 (ACS6). Moreover, ethylene signaling contributes to OG‐triggered immunity activation, and lack of CPK5, CPK6 and CPK11 affects the duration of OG‐ and flg22‐induced gene expression, indicating that these kinases are shared elements of both DAMP and MAMP signaling pathways.     

Separation of early and late responses to herbivory in Arabidopsis by changing plasmodesmal function

Herbivory results in an array of physiological changes in the host that are separable from the associated physical damage. We have made the surprising observation that an Arabidopsis line (pdko3) mutated in genes encoding plasmodesmal proteins is defective in some, but not all, of the typical plant responses to herbivory. We tested the responses of plasma transmembrane potential (Vm) depolarization, voltage gated K⁺ channel activity, cytosolic calcium [Ca²⁺]_cyt and reactive oxygen species (ROS) (H₂O₂ and NO) release, shoot‐to‐root signaling, biosynthesis of the phytohormone jasmonic acid (JA) and the elicitation of volatile organic compounds (VOCs). Following herbivory and the release of factors present in insect oral secretions (including a putative β‐galactofuranose polysaccharide), both the pdko3 and wild type (WT) plants showed a increased accumulation of [Ca²⁺]_cyt, NO and H₂O₂. In contrast, unlike WT plants, the mutant line showed an almost complete loss of voltage gated K⁺ channel activity and Vm depolarization, a loss of shoot‐induced root‐Vm depolarization, a loss of activation and regulation of gene expression of the JA defense pathway, and a much diminished release and altered profile of VOCs. The mutations in genes for plasmodesmal proteins have provided valuable genetic tools for the dissection of the complex spectrum of responses to herbivory and shown us that the responses to herbivory can be separated into a calcium‐activated oxidative response and a K⁺‐dependent Vm‐activated jasmonate response associated with the release of VOCs.     

The Arabidopsis heterotrimeric G‐protein β subunit,AGB1, is required for guard cell calcium sensing and calcium‐induced calcium release

Cytosolic calcium concentration ([Ca²⁺]_cyt) and heterotrimeric G‐proteins are universal eukaryotic signaling elements. In plant guard cells, extracellular calcium (Ca_o) is as strong a stimulus for stomatal closure as the phytohormone abscisic acid (ABA), but underlying mechanisms remain elusive. Here, we report that the sole Arabidopsis heterotrimeric Gβ subunit, AGB1, is required for four guard cell Ca_o responses: induction of stomatal closure; inhibition of stomatal opening; [Ca²⁺]_cyt oscillation; and inositol 1,4,5‐trisphosphate (InsP3) production. Stomata in wild‐type Arabidopsis (Col) and in mutants of the canonical Gα subunit, GPA1, showed inhibition of stomatal opening and promotion of stomatal closure by Ca_o. By contrast, stomatal movements of agb1 mutants and agb1/gpa1 double‐mutants, as well as those of the agg1agg2 Gγ double‐mutant, were insensitive to Ca_o. These behaviors contrast with ABA‐regulated stomatal movements, which involve GPA1 and AGB1/AGG3 dimers, illustrating differential partitioning of G‐protein subunits among stimuli with similar ultimate impacts, which may facilitate stimulus‐specific encoding. AGB1 knockouts retained reactive oxygen species and NO production, but lost YC3.6‐detected [Ca²⁺]_cyt oscillations in response to Ca_o, initiating only a single [Ca²⁺]_cyt spike. Experimentally imposed [Ca²⁺]_cyt oscillations restored stomatal closure in agb1. Yeast two‐hybrid and bimolecular complementation fluorescence experiments revealed that AGB1 interacts with phospholipase Cs (PLCs), and Ca_o induced InsP3 production in Col but not in agb1. In sum, G‐protein signaling via AGB1/AGG1/AGG2 is essential for Ca_o‐regulation of stomatal apertures, and stomatal movements in response to Ca_o apparently require Ca²⁺‐induced Ca²⁺ release that is likely dependent on Gβγ interaction with PLCs leading to InsP3 production.     

DOWNY MILDEW RESISTANT 6 and DMR6‐LIKE OXYGENASE 1 are partially redundant but distinct suppressors of immunity in Arabidopsis

Arabidopsis downy mildew resistant 6 (dmr6) mutants have lost their susceptibility to the downy mildew Hyaloperonospora arabidopsidis. Here we show that dmr6 is also resistant to the bacterium Pseudomonas syringae and the oomycete Phytophthora capsici. Resistance is accompanied by enhanced defense gene expression and elevated salicylic acid levels. The suppressive effect of the DMR6 oxygenase was confirmed in transgenic Arabidopsis lines overexpressing DMR6 that show enhanced susceptibility to H. arabidopsidis, P. capsici, and P. syringae. Phylogenetic analysis of the superfamily of 2‐oxoglutarate Fe(II)‐dependent oxygenases revealed a subgroup of DMR6‐LIKE OXYGENASEs (DLOs). Within Arabidopsis, DMR6 is most closely related to DLO1 and DLO2. Overexpression of DLO1 and DLO2 in the dmr6 mutant restored the susceptibility to downy mildew indicating that DLOs negatively affect defense, similar to DMR6. DLO1, but not DLO2, is co‐expressed with DMR6, showing strong activation during pathogen attack and following salicylic acid treatment. DMR6 and DLO1 differ in their spatial expression pattern in downy mildew‐infected Arabidopsis leaves; DMR6 is mostly expressed in cells that are in contact with hyphae and haustoria of H. arabidopsidis, while DLO1 is expressed mainly in the vascular tissues near infection sites. Strikingly, the dmr6‐3_dlo1 double mutant, that is completely resistant to H. arabidopsidis, showed a strong growth reduction that was associated with high levels of salicylic acid. We conclude that DMR6 and DLO1 redundantly suppress plant immunity, but also have distinct activities based on their differential localization of expression.     

Functional analysis of chimeric lysin motif domain receptors mediating Nod factor‐induced defense signaling in Arabidopsis thaliana and chitin‐induced nodulation signaling in Lotus japonicus

The expression of chimeric receptors in plants is a way to activate specific signaling pathways by corresponding signal molecules. Defense signaling induced by chitin from pathogens and nodulation signaling of legumes induced by rhizobial Nod factors (NFs) depend on receptors with extracellular lysin motif (LysM) domains. Here, we constructed chimeras by replacing the ectodomain of chitin elicitor receptor kinase 1 (AtCERK1) of Arabidopsis thaliana with ectodomains of NF receptors of Lotus japonicus (LjNFR1 and LjNFR5). The hybrid constructs, named LjNFR1–AtCERK1 and LjNFR5–AtCERK1, were expressed in cerk1‐2, an A. thaliana CERK1 mutant lacking chitin‐induced defense signaling. When treated with NFs from Rhizobium sp. NGR234, cerk1‐2 expressing both chimeras accumulated reactive oxygen species, expressed chitin‐responsive defense genes and showed increased resistance to Fusarium oxysporum. In contrast, expression of a single chimera showed no effects. Likewise, the ectodomains of LjNFR1 and LjNFR5 were replaced by those of OsCERK1 (Oryza sativa chitin elicitor receptor kinase 1) and OsCEBiP (O. sativa chitin elicitor‐binding protein), respectively. The chimeras, named OsCERK1–LjNFR1 and OsCEBiP–LjNFR5, were expressed in L. japonicus NF receptor mutants (nfr1‐1; nfr5‐2) carrying a GUS (β‐glucuronidase) gene under the control of the NIN (nodule inception) promoter. Upon chitin treatment, GUS activation reflecting nodulation signaling was observed in the roots of NF receptor mutants expressing both chimeras, whereas a single construct was not sufficient for activation. Hence, replacement of ectodomains in LysM domain receptors provides a way to specifically trigger NF‐induced defense signaling in non‐legumes and chitin‐induced nodulation signaling in legumes.     

Arabidopsis thaliana glutamate receptor ion channel function demonstrated by ion pore transplantation

Ionotropic glutamate receptors (iGluRs) are ligand-gated cation channels that mediate fast excitatory neurotransmission in the mammalian central nervous system. In the model plant Arabidopsis thaliana, a large family of 20 genes encoding proteins that share similarities with animal iGluRs in sequence and predicted secondary structure has been discovered. Members of this family, termed AtGLRs (A. thaliana glutamate receptors), have been implicated in root development, ion transport, and several metabolic and signalling pathways. However, there is still no direct proof of ligand-gated ion channel function of any AtGLR subunit. We used a domain transplantation technique to directly test whether the putative ion pore domains of AtGLRs can conduct ions. To this end, we transplanted the ion pore domains of 17 AtGLR subunits into rat α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (GluR1) and kainate (GluR6) receptor subunits and tested the resulting chimaeras for ion channel function in the Xenopus oocyte expression system. We show that AtGLR1.1 and AtGLR1.4 have functional Na⁺-, K⁺-, and Ca²⁺-permeable ion pore domains. The properties of currents through the AtGLR1.1 ion pore match those of glutamate-activated currents, depolarisations, and glutamate-triggered Ca²⁺ influxes observed in plant cells. We conclude that some AtGLRs have functional non-selective cation pores.     

Annexin 1 regulates the H2O2‐induced calcium signature in Arabidopsis thaliana roots

Hydrogen peroxide is the most stable of the reactive oxygen species (ROS) and is a regulator of development, immunity and adaptation to stress. It frequently acts by elevating cytosolic free Ca²⁺ ([Ca²⁺]_cyt) as a second messenger, with activation of plasma membrane Ca²⁺‐permeable influx channels as a fundamental part of this process. At the genetic level, to date only the Ca²⁺‐permeable Stelar K⁺ Outward Rectifier (SKOR) channel has been identified as being responsive to hydrogen peroxide. We show here that the ROS‐regulated Ca²⁺ transport protein Annexin 1 in Arabidopsis thaliana (AtANN1) is involved in regulating the root epidermal [Ca²⁺]_cyt response to stress levels of extracellular hydrogen peroxide. Peroxide‐stimulated [Ca²⁺]_cyt elevation (determined using aequorin luminometry) was aberrant in roots and root epidermal protoplasts of the Atann1 knockout mutant. Similarly, peroxide‐stimulated net Ca²⁺ influx and K⁺ efflux were aberrant in Atann1 root mature epidermis, determined using extracellular vibrating ion‐selective microelectrodes. Peroxide induction of GSTU1 (Glutathione‐S‐Transferase1 Tau 1), which is known to be [Ca²⁺]_cyt‐dependent was impaired in mutant roots, consistent with a lesion in signalling. Expression of AtANN1 in roots was suppressed by peroxide, consistent with the need to restrict further Ca²⁺ influx. Differential regulation of annexin expression was evident, with AtANN2 down‐regulation but up‐regulation of AtANN3 and AtANN4. Overall the results point to involvement of AtANN1 in shaping the root peroxide‐induced [Ca²⁺]_cyt signature and downstream signalling.     

Pepper pathogenesis‐related protein 4c is a plasma membrane‐localized cysteine protease inhibitor that is required for plant cell death and defense signaling

Xanthomonas campestris pv. vesicatoria (Xcv) type III effector AvrBsT triggers programmed cell death (PCD) and activates the hypersensitive response (HR) in plants. Here, we isolated and identified the plasma membrane localized pathogenesis‐related (PR) protein 4c gene (CaPR4c) from pepper (Capsicum annuum) leaves undergoing AvrBsT‐triggered HR cell death. CaPR4c encodes a protein with a signal peptide and a Barwin domain. Recombinant CaPR4c protein expressed in Escherichia coli exhibited cysteine protease‐inhibitor activity and ribonuclease (RNase) activity. Subcellular localization analyses revealed that CaPR4c localized to the plasma membrane in plant cells. CaPR4c expression was rapidly and specifically induced by avirulent Xcv (avrBsT) infection. Transient expression of CaPR4c caused HR cell death in pepper leaves, which was accompanied by enhanced accumulation of H₂O₂ and significant induction of some defense‐response genes. Deletion of the signal peptide from CaPR4c abolished the induction of HR cell death, indicating a requirement for plasma membrane localization of CaPR4c for HR cell death. CaPR4c silencing in pepper disrupted both basal and AvrBsT‐triggered resistance responses, and enabled Xcv proliferation in infected leaves. H₂O₂ accumulation, cell‐death induction, and defense‐response gene expression were distinctly reduced in CaPR4c‐silenced pepper. CaPR4c overexpression in transgenic Arabidopsis plants conferred greater resistance against infection by Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis. These results collectively suggest that CaPR4c plays an important role in plant cell death and defense signaling.     

A critical role for Arabidopsis MILDEW RESISTANCE LOCUS O2 in systemic acquired resistance

Members of the MILDEW RESISTANCE LOCUS O (MLO) gene family confer susceptibility to powdery mildews in different plant species, and their existence therefore seems to be disadvantageous for the plant. We recognized that expression of the Arabidopsis MLO2 gene is induced after inoculation with the bacterial pathogen Pseudomonas syringae, promoted by salicylic acid (SA) signaling, and systemically enhanced in the foliage of plants exhibiting systemic acquired resistance (SAR). Importantly, distinct mlo2 mutant lines were unable to systemically increase resistance to bacterial infection after inoculation with P. syringae, indicating that the function of MLO2 is necessary for biologically induced SAR in Arabidopsis. Our data also suggest that the close homolog MLO6 has a supportive but less critical role in SAR. In contrast to SAR, basal resistance to bacterial infection was not affected in mlo2. Remarkably, SAR‐defective mlo2 mutants were still competent in systemically increasing the levels of the SAR‐activating metabolites pipecolic acid (Pip) and SA after inoculation, and to enhance SAR‐related gene expression in distal plant parts. Furthermore, although MLO2 was not required for SA‐ or Pip‐inducible defense gene expression, it was essential for the proper induction of disease resistance by both SAR signals. We conclude that MLO2 acts as a critical downstream component in the execution of SAR to bacterial infection, being required for the translation of elevated defense responses into disease resistance. Moreover, our data suggest a function for MLO2 in the activation of plant defense priming during challenge by P. syringae.     

Long‐chain base phosphates modulate pollen tube growth via channel‐mediated influx of calcium

Long‐chain base phosphates (LCBPs) have been correlated with amounts of crucial biological processes ranging from cell proliferation to apoptosis in animals. However, their functions in plants remain largely unknown. Here, we report that LCBPs, sphingosine‐1‐phosphate (S1P) and phytosphingosine‐1‐phosphate (Phyto‐S1P), modulate pollen tube growth in a concentration‐dependent bi‐phasic manner. The pollen tube growth in the stylar transmitting tissue was promoted by SPHK1 overexpression (SPHK1‐OE) but dampened by SPHK1 knockdown (SPHK1‐KD) compared with wild‐type of Arabidopsis; however, there was no detectable effect on in vitro pollen tube growth caused by misexpression of SPHK1. Interestingly, exogenous S1P or Phyto‐S1P applications could increase the pollen tube growth rate in SPHK1‐OE, SPHK1‐KD and wild‐type of Arabidopsis. Calcium ion (Ca²⁺)‐imaging analysis showed that S1P triggered a remarkable increase in cytosolic Ca²⁺ concentration in pollen. Extracellular S1P induced hyperpolarization‐activated Ca²⁺ currents in the pollen plasma membrane, and the Ca²⁺ current activation was mediated by heterotrimeric G proteins. Moreover, the S1P‐induced increase of cytosolic free Ca²⁺ inhibited the influx of potassium ions in pollen tubes. Our findings suggest that LCBPs functions in a signaling cascade that facilitates Ca²⁺ influx and modulates pollen tube growth.     

Engineered silver nanoparticles are sensed at the plasma membrane and dramatically modify the physiology of Arabidopsis thaliana plants

Silver nanoparticles (Ag NPs) are the world's most important nanomaterial and nanotoxicant. The aim of this study was to determine the early stages of interactions between Ag NPs and plant cells, and to investigate their physiological roles. We have shown that the addition of Ag NPs to cultivation medium, at levels above 300 mg L^?1, inhibited Arabidopsis thaliana root elongation and leaf expansion. This also resulted in decreased photosynthetic efficiency and the extreme accumulation of Ag in tissues. Acute application of Ag NPs induced a transient elevation of [Ca²⁺]_cyt and the accumulation of reactive oxygen species (ROS; partially generated by NADPH oxidase). Whole‐cell patch‐clamp measurements on root cell protoplasts demonstrated that Ag NPs slightly inhibited plasma membrane K⁺ efflux and Ca²⁺ influx currents, or caused membrane breakdown; however, in excised outside‐out patches, Ag NPs activated Gd³⁺‐sensitive Ca²⁺ influx channels with unitary conductance of approximately 56 pS. Bulk particles did not modify the plasma membrane currents. Tests with electron paramagnetic resonance spectroscopy showed that Ag NPs were not able to catalyse hydroxyl radical generation, but that they directly oxidized the major plant antioxidant, l ‐ascorbic acid. Overall, the data presented shed light on mechanisms of the impact of nanosilver on plant cells, and show that these include the induction of classical stress signalling reactions (mediated by [Ca²⁺]_cyt and ROS) and a specific effect on the plasma membrane conductance and the reduced ascorbate.     

Metabotropic glutamate receptor 6 signaling enhances TRPM1 calcium channel function and increases melanin content in human melanocytes

Mutations in TRPM1, a calcium channel expressed in retinal bipolar cells and epidermal melanocytes, cause complete congenital stationary night blindness with no discernible skin phenotype. In the retina, TRPM1 activity is negatively coupled to metabotropic glutamate receptor 6 (mGluR6) signaling through Gα_o and TRPM1 mutations result in the loss of responsiveness of TRPM1 to mGluR6 signaling. Here, we show that human melanocytes express mGluR6, and treatment of melanocytes with L‐AP4, a type III mGluR‐selective agonist, enhances Ca²⁺ uptake. Knockdown of TRPM1 or mGluR6 by shRNA abolished L‐AP4‐induced Ca²⁺ influx and TRPM1 currents, showing that TRPM1 activity in melanocytes is positively coupled to mGluR6 signaling. Gα_o protein is absent in melanocytes. However, forced expression of Gα_o restored negative coupling of TRPM1 to mGluR6 signaling, but treatment with pertussis toxin, an inhibitor of G_i/G_o proteins, did not affect basal or mGluR6‐induced Ca²⁺ uptake. Additionally, chronic stimulation of mGluR6 altered melanocyte morphology and increased melanin content. These data suggest differences in coupling of TRPM1 function to mGluR6 signaling explain different cellular responses to glutamate in the retina and the skin.     

Alternative Splicing-Mediated Targeting of the Arabidopsis GLUTAMATE RECEPTOR3.5 to Mitochondria Affects Organelle Morphology

Since the discovery of 20 genes encoding for putative ionotropic glutamate receptors in the Arabidopsis (Arabidopsis thaliana) genome, there has been considerable interest in uncovering their physiological functions. For many of these receptors, neither their channel formation and/or physiological roles nor their localization within the plant cells is known. Here, we provide, to our knowledge, new information about in vivo protein localization and give insight into the biological roles of the so-far uncharacterized Arabidopsis GLUTAMATE RECEPTOR3.5 (AtGLR3.5), a member of subfamily 3 of plant glutamate receptors. Using the pGREAT vector designed for the expression of fusion proteins in plants, we show that a splicing variant of AtGLR3.5 targets the inner mitochondrial membrane, while the other variant localizes to chloroplasts. Mitochondria of knockout or silenced plants showed a strikingly altered ultrastructure, lack of cristae, and swelling. Furthermore, using a genetically encoded mitochondria-targeted calcium probe, we measured a slightly reduced mitochondrial calcium uptake capacity in the knockout mutant. These observations indicate a functional expression of AtGLR3.5 in this organelle. Furthermore, AtGLR3.5-less mutant plants undergo anticipated senescence. Our data thus represent, to our knowledge, the first evidence of splicing-regulated organellar targeting of a plant ion channel and identify the first cation channel in plant mitochondria from a molecular point of view.In vertebrates, ionotropic glutamate receptors (iGluRs in animals) are ligand-gated cation channels that mediate the majority of the excitatory neurotransmission in the central nervous system (Dingledine et al., 1999). In the model plant Arabidopsis (Arabidopsis thaliana), 20 genes encoding homologs of animal iGluRs have been identified (Lam et al., 1998). According to phylogenetic analyses, the Arabidopsis GLUTAMATE RECEPTOR (AtGLR) homologs can be subdivided into three separate subgroups (Chiu et al., 1999, 2002). Some evidence for the channel-forming ability by plant ionotropic glutamate receptors (iGLRs) has been obtained only recently, and only for AtGLR3.4 and AtGLR1.4 expressed in heterologous systems (Vincill et al., 2012; Tapken et al., 2013). Studies with transgenic plants suggested roles of members of the plant GLR family in Ca²⁺ fluxes (AtGLR2; Kim et al., 2001), coordination of mitotic activity in the root apical meristem (Li et al., 2006), regulation of abscisic acid biosynthesis and water balance (AtGLR1.1; Kang and Turano, 2003; Kang et al., 2004), carbon/nitrogen sensing (AtGLR1.1; Kang and Turano, 2003), resistance against fungal infection (Kang et al., 2006), leaf-to-leaf wound signaling (Mousavi et al., 2013), and lateral root initiation (Vincill et al., 2013). Application of antagonists and agonists of animal iGluRs revealed that plant GLRs might be involved in the regulation of root growth and branching (Walch-Liu et al., 2006), in light signal transduction (Lam et al., 1998), and in the response to aluminum (Sivaguru et al., 2003). In various plant cell types, the agonists Glu- and Gly-induced plasma membrane depolarization and a rise in intracellular Ca²⁺ concentration that were inhibited by blockers of nonselective cation channels (NSCCs) and by antagonists of animal iGluRs (Dennison and Spalding, 2000; Dubos et al., 2003; Meyerhoff et al., 2005; Krol et al., 2007; Kwaaitaal et al., 2011; Michard et al., 2011). Furthermore, Glu-activated cation currents in patch-clamped root protoplasts were inhibited by NSCC blockers such as La³⁺ and Gd³⁺ (Demidchik et al., 2004). Therefore, it was proposed that plant iGLRs can form Ca²⁺-permeable NSCCs, are inhibited by animal iGluR antagonists, and might contribute to the shaping of plant Ca²⁺ signaling (McAinsh and Pittman, 2009). Studies using AtGLR3.3 mutant plants showed that intracellular Ca²⁺ rise and membrane depolarization induced by Glu in Arabidopsis hypocotyls and root cells are correlated with the presence of AtGLR3.3 (Qi et al., 2006; Stephens et al., 2008).However, most plant iGLRs, when expressed in heterologous systems, do not give rise to any current (e.g. in Xenopus spp. oocytes) or are toxic to host cells (e.g. in mammalian cells; Davenport, 2002). Recently, to examine whether AtGLR homologs possess functional ion channel domains, Tapken and Hollmann (2008) transplanted the pore loop together with two adjacent intracellular loops of 17 AtGLR subunits into two rat iGluR subunits and tested the resulting chimeric receptors for ion channel activity in the heterologous expression system Xenopus spp. oocyte. They showed that AtGLR1.1 and AtGLR1.4 have functional ion pore domains. The AtGLR1.1 pores are permeable to Na⁺, K⁺, and Ca²⁺ and are blocked by the nonspecific cation channel blocker La³⁺ (Tapken and Hollmann, 2008). Recent work has demonstrated that the expression of full-length AtGLR1.4 in oocytes gives rise to an amino acid-activated, nonselective, calcium-permeable channel that was found to be inhibited by the animal iGluR modulators 6,7-dinitroquinoxaline-2,3-dione and 6-cyano-7-nitroquinoxaline-2,3-dione (Tapken et al., 2013).The study of these channels has so far been restricted to those members that are located in the plasma membrane and were proved to be functional in the expression systems used. Instead, various localization prediction tools suggest that some of the plant GLRs might have chloroplast and mitochondrial targeting. In general, determining the subcellular localization of a protein is an important step toward understanding its function. We recently reported the localization of GLR3.4 to the inner chloroplast membrane (Teardo et al., 2011), which was also shown to harbor a 6,7-dinitroquinoxaline-2,3-dione-sensitive, calcium-permeable channel activity (Teardo et al., 2010). No other studies have addressed the eventual subcellular localization of other putative Glu receptors.In this work, we show that an isoform of GLR3.5 is efficiently targeted to the mitochondria. Functional expression of the channel in this organelle is indicated by the fact that its absence in knockout plants leads to a dramatically altered ultrastructure of mitochondria that impacts the plant physiology, ultimately leading to an anticipated senescence.     

Reactive oxygen species generated in chloroplasts contribute to tobacco leaf infection by the necrotrophic fungus Botrytis cinerea

Reactive oxygen species (ROS) play fundamental roles in plant responses to pathogen infection, including modulation of cell death processes and defense‐related gene expression. Cell death triggered as part of the hypersensitive response enhances resistance to biotrophic pathogens, but favors the virulence of necrotrophs. Even though the involvement of ROS in the orchestration of defense responses is well established, the relative contribution of specific subcellular ROS sources to plant resistance against microorganisms with different pathogenesis strategies is not completely known. The aim of this work was to investigate the role of chloroplastic ROS in plant defense against a typical necrotrophic fungus, Botrytis cinerea. For this purpose, we used transgenic Nicotiana tabacum (tobacco) lines expressing a plastid‐targeted cyanobacterial flavodoxin (pfld lines), which accumulate lower chloroplastic ROS in response to different stresses. Tissue damage and fungal growth were significantly reduced in infected leaves of pfld plants, as compared with infected wild‐type (WT) counterparts. ROS build‐up triggered by Botrytis infection and associated with chloroplasts was significantly decreased (70–80%) in pfld leaves relative to the wild type. Phytoalexin accumulation and expression of pathogenesis‐related genes were induced to a lower degree in pfld plants than in WT siblings. The impact of fungal infection on photosynthetic activity was also lower in pfld leaves. The results indicate that chloroplast‐generated ROS play a major role in lesion development during Botrytis infection. This work demonstrates that the modulation of chloroplastic ROS levels by the expression of a heterologous antioxidant protein can provide a significant degree of protection against a canonical necrotrophic fungus.     

Homologous RXLR effectors from Hyaloperonospora arabidopsidis and Phytophthora sojae suppress immunity in distantly related plants

Diverse pathogens secrete effector proteins into plant cells to manipulate host cellular processes. Oomycete pathogens contain large complements of predicted effector genes defined by an RXLR host cell entry motif. The genome of Hyaloperonospora arabidopsidis (Hpa, downy mildew of Arabidopsis) contains at least 134 candidate RXLR effector genes. Only a small subset of these genes is conserved in related oomycetes from the Phytophthora genus. Here, we describe a comparative functional characterization of the Hpa RXLR effector gene HaRxL96 and a homologous gene, PsAvh163, from the Glycine max (soybean) pathogen Phytophthora sojae. HaRxL96 and PsAvh163 are induced during the early stages of infection and carry a functional RXLR motif that is sufficient for protein uptake into plant cells. Both effectors can suppress immune responses in soybean. HaRxL96 suppresses immunity in Nicotiana benthamiana, whereas PsAvh163 induces an HR‐like cell death response in Nicotiana that is dependent on RAR1 and Hsp90.1. Transgenic Arabidopsis plants expressing HaRxL96 or PsAvh163 exhibit elevated susceptibility to virulent and avirulent Hpa, as well as decreased callose deposition in response to non‐pathogenic Pseudomonas syringae. Both effectors interfere with defense marker gene induction, but do not affect salicylic acid biosynthesis. Together, these experiments demonstrate that evolutionarily conserved effectors from different oomycete species can suppress immunity in plant species that are divergent from the source pathogen’s host.     

Four Arabidopsis berberine bridge enzyme‐like proteins are specific oxidases that inactivate the elicitor‐active oligogalacturonides

Recognition of endogenous molecules acting as ‘damage‐associated molecular patterns’ (DAMPs) is a key feature of immunity in both animals and plants. Oligogalacturonides (OGs), i.e. fragments derived from the hydrolysis of homogalacturonan, a major component of pectin are a well known class of DAMPs that activate immunity and protect plants against several microbes. However, hyper‐accumulation of OGs severely affects growth, eventually leading to cell death and clearly pointing to OGs as players in the growth‐defence trade‐off. Here we report a mechanism that may control the homeostasis of OGs avoiding their deleterious hyper‐accumulation. By combining affinity chromatography on acrylamide‐trapped OGs and other procedures, an Arabidopsis thaliana enzyme that specifically oxidizes OGs was purified and identified. The enzyme was named OG OXIDASE 1 (OGOX1) and shown to be encoded by the gene At4g20830. As a typical flavo‐protein, OGOX1 is a sulphite‐sensitive H₂O₂‐producing enzyme that displays maximal activity on OGs with a degree of polymerization >4. OGOX1 belongs to a large gene family of mainly apoplastic putative FAD‐binding proteins [Berberine Bridge Enzyme‐like (BBE‐like); 27 members], whose biochemical and biological function is largely unexplored. We have found that at least four BBE‐like enzymes in Arabidopsis are OG oxidases (OGOX1–4). Oxidized OGs display a reduced capability of activating the immune responses and are less hydrolysable by fungal polygalacturonases. Plants overexpressing OGOX1 are more resistant to Botrytis cinerea, pointing to a crucial role of OGOX enzymes in plant immunity.