Bipyridines mediate electron transfer from an electrode to nicotinamide adenine dinucleotide phosphate.

Biocatalysts are widely used in industry, but few examples of the use of oxidoreductases, in which enzymatic function often requires electrons, have been reported. NADPH is a cofactor that supplies an electron to oxidoreductases, but is consequently inactivated and no longer able to act as an electron donor. NADP+ can not receive electrons from electrodes through straightforward electrochemistry owing to its complicated three-dimensional structure. This study reports that bipyridines effectively mediate electron transfer between an electrode and NADP+, allowing them to serve as electron mediators for NADPH production. Using bipyridines, quinones, and anilines, which have negative oxidation-reduction potentials, an electrochemical investigation was conducted into whether electrons were transferred to NADP+. Only bipyridines with a reduction potential near -1.0 V exhibited electron transfer. Furthermore, the NADPH production level was measured using spectroscopy. NADPH was efficiently produced using bipyridines, such as methyl viologen and ethyl viologen, in which the bipyridyl 1- and 1'-positions bear small substituents. However, methyl viologen caused a dehydrogenation reaction of NADPH, making it unsuitable as an electron mediator for NADPH production. The dehydrogenation reaction did not occur using ethyl viologen. These results indicated that NADP+ can be reduced more effectively using substituents that prevent a dehydrogenation reaction at the bipyridyl 1- and 1'-positions while maintaining the reducing power.

How do Plants Keep their Functional Integrity?

Unlike animals, plants possess a non-strict and sometimes very fuzzy morphology. Mutual proportions of plant parts can vary to a much greater extent than in animals, changing according to the environmental conditions and the plant needs of nutrients, water and light. Despite the existence of this fundamental difference between plants and animals, it passes almost non-reflected in most studies on plants. In this review we make a preliminary attempt to gather together the mechanisms by which plants preserve their integrity, not loosing at the same time the physiological (and morphological) flexibility which allows them adapting to the different environments they can populate.

Measurement of the Caspase-1-Like Activity of Vacuolar Processing Enzyme in Plants.

Caspase-like activities are essential to regulate programed cell death in plants. Although no caspase orthologous enzymes with aspartic acid specificity have been identified in plants, vacuolar processing enzyme (VPE) exhibits a caspase-1-like activity. In this chapter, we introduce two methods for the measurement of the caspase-1-like/VPE activity. These methods are based on the cleavage of caspase-1 specific synthetic substrates and on monitoring the active forms of VPE using a biotinylated-inhibitor blot analysis. Both methods are also adaptable to other plant caspase-like activities.

WRKY70 prevents axenic activation of plant immunity by direct repression of SARD1.

SARD1 is an activator of plant immunity that promotes production of the hormone salicylic acid (SA) and activation of defense gene expression. SARD1 itself is strongly inducible by infection. Here, we investigated the transcriptional control of SARD1. We used yeast one-hybrid assays to identify WRKY70. The WRKY70 binding site was defined using electrophoretic mobility shift assays, and its importance was investigated using an Arabidopsis thaliana protoplast system. The effect of wrky70 mutations was studied by measurements of pathogen growth, SA concentrations, and gene expression by RNA-seq. WRKY70 binds to a GACTTTT motif in the SARD1 promoter in yeast and Arabidopsis protoplasts. Plants with wrky70 mutations have elevated expression of SARD1 in the absence of pathogens, but not when infected. Expression profiling revealed that WRKY70 represses many pathogen-inducible genes in the absence of pathogens, yet is required for activation of many other pathogen-inducible genes in infected plants. The GACTTTT motif is enriched in the promoters of both these gene sets, and conserved in SARD1 orthologs within the Brassicaceae. WRKY70 represses SARD1 by binding the motif GACTTTT in the absence of pathogens. Conservation of the WRKY70 binding among the Brassicaceae suggests that WRKY70 repression of SARD1 is important for fitness.

Quantification of Plant Cell Death by Electrolyte Leakage Assay.

We describe a protocol to measure the electrolyte leakage from plant tissues, resulting from loss of cell membrane integrity, which is a common definition of cell death. This simple protocol is designed to measure the electrolyte leakage from a tissue sample over a time course, so that the extent of cell death in the tissue can be monitored dynamically. In addition, it is easy to handle many tissue samples in parallel, which allows a high level of biological replication. Although the protocol is exemplified by cell death in Arabidopsis in response to pathogen challenge, it is easily applicable to other types of plant cell death.

Involvement of Adapter Protein Complex 4 in Hypersensitive Cell Death Induced by Avirulent Bacteria.

Plant immunity to avirulent bacterial pathogens is associated with subcellular membrane dynamics including fusion between the vacuolar and plasma membranes, resulting in hypersensitive cell death. Here, we report that ADAPTOR PROTEIN COMPLEX-4 (AP-4) subunits are involved in plant immunity associated with hypersensitive cell death. We isolated a mutant with a defect in resistance to an avirulent strain of Pseudomonas syringae pv. tomato (Pto) DC3000 avrRpm1 from a vacuolar protein sorting mutant library of Arabidopsis (Arabidopsis thaliana). The mutant was identical to gfs4-1, which has a mutation in the gene encoding the AP-4 subunit AP4B. Thus, we focused on AP4B and another subunit, AP4E. All of the mutants (ap4b-3, ap4b-4, ap4e-1, and ap4e-2) were defective in hypersensitive cell death and resistance to Pto DC3000 with the type III effector AvrRpm1 or AvrRpt2, both of which are recognized on the plasma membrane, while they showed slightly enhanced susceptibility to the type-III-secretion-deficient P. syringae strain hrcC On the other hand, both ap4b-3 and ap4b-4 showed no defect in resistance to Pto DC3000 with the type III effector AvrRps4, which is recognized in the cytosol and does not induce hypersensitive cell death. Upon infection with Pto DC3000 avrRpt2, the ap4b-3 and ap4b-4 leaf cells did not show fusion between vacuolar and plasma membranes, whereas the wild-type leaf cells did. These results suggest that AP-4 contributes to cell death-associated immunity, possibly via membrane fusion, after type III effector-recognition on the plasma membrane.

A plant effector-triggered immunity signaling sector is inhibited by pattern-triggered immunity.

Since signaling machineries for two modes of plant-induced immunity, pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), extensively overlap, PTI and ETI signaling likely interact. In an Arabidopsis quadruple mutant, in which four major sectors of the signaling network, jasmonate, ethylene, PAD4, and salicylate, are disabled, the hypersensitive response (HR) typical of ETI is abolished when the Pseudomonas syringae effector AvrRpt2 is bacterially delivered but is intact when AvrRpt2 is directly expressed in planta These observations led us to discovery of a network-buffered signaling mechanism that mediates HR signaling and is strongly inhibited by PTI signaling. We named this mechanism the ETI-Mediating and PTI-Inhibited Sector (EMPIS). The signaling kinetics of EMPIS explain apparently different plant genetic requirements for ETI triggered by different effectors without postulating different signaling machineries. The properties of EMPIS suggest that information about efficacy of the early immune response is fed back to the immune signaling network, modulating its activity and limiting the fitness cost of unnecessary immune responses.

Nup82 functions redundantly with Nup136 in a salicylic acid-dependent defense response of Arabidopsis thaliana.

The nuclear pore complex (NPC) comprises more than 30 nucleoporins (Nups). NPC mediates macromolecular trafficking between the nucleoplasm and the cytoplasm, but specific roles of individual Nups are poorly understood in higher plants. Here, we show that the novel nucleoporin unique to angiosperm plants (designated as Nup82) functions in a salicylic acid-dependent defense in a redundant manner with Nup136, which is a component of the nuclear basket in the NPC. Arabidopsis thaliana Nup82 had a similar amino acid sequence to the N-terminal half of Nup136 and a Nup82-GFP fusion was localized on the nuclear envelope. Immunoprecipitation and bimolecular fluorescence complementation analyses revealed that Nup82 interacts with the NPC components Nup136 and RAE1. The double knockout mutant nup82 nup136 showed severe growth defects, while the single knockout mutant nup82 did not, suggesting that Nup82 functions redundantly with Nup136. nup82 nup136 impaired benzothiadiazole (an analog of salicylic acid)-induced resistance to the virulent bacteria Pseudomonas syringae pv. tomato DC3000. Furthermore, transcriptome analysis of nup82 nup136 indicates that deficiency of Nup82 and Nup136 causes noticeable downregulation of immune-related genes. These results suggest that Nup82 and Nup136 are redundantly involved in transcriptional regulation of salicylic acid-responsive genes through nuclear transport of signaling molecules.

Vacuolar convolution: possible mechanisms and role of phosphatidylinositol 3,5-bisphosphate.

The central or lytic vacuole is the largest intracellular organelle in plant cells, but we know unacceptably little about the mechanisms regulating its function in vivo. The underlying reasons are related to difficulties in accessing this organelle without disrupting the cellular integrity and to the dynamic morphology of the vacuole, which lacks a defined structure. Among such morphological changes, vacuolar convolution is probably the most commonly observed event, reflected in the (reversible) transformation of a large central vacuole into a structure consisting of interconnected bubbles of a smaller size. Such behaviour is observed in plant cells subjected to hyperosmotic stress but also takes place in physiological conditions (e.g. during stomatal closure). Although vacuolar convolution is a relatively common phenomenon in plants, studies aimed at elucidating its execution mechanisms are rather scarce. In the present review, we analyse the available evidence on the participation of the cellular cytoskeleton and ion transporters in vacuolar morphology dynamics, putting special emphasis on the available evidence of the role played by phosphatidylinositol 3,5-bisphosphate in this process.

Hexose Oxidase-Mediated Hydrogen Peroxide as a Mechanism for the Antibacterial Activity in the Red Seaweed Ptilophora subcostata.

Marine algae have unique defense strategies against microbial infection. However, their mechanisms of immunity remain to be elucidated and little is known about the similarity of the immune systems of marine algae and terrestrial higher plants. Here, we suggest a possible mechanism underlying algal immunity, which involves hexose oxidase (HOX)-dependent production of hydrogen peroxide (H2O2). We examined crude extracts from five different red algal species for their ability to prevent bacterial growth. The extract from one of these algae, Ptilophora subcostata, was particularly active and prevented the growth of gram-positive and -negative bacteria, which was completely inhibited by treatment with catalase. The extract did not affect the growth of either a yeast or a filamentous fungus. We partially purified from P. subcostata an enzyme involved in its antibacterial activity, which shared 50% homology with the HOX of red seaweed Chondrus crispus. In-gel carbohydrate oxidase assays revealed that P. subcostata extract had the ability to produce H2O2 in a hexose-dependent manner and this activity was highest in the presence of galactose. In addition, Bacillus subtilis growth was strongly suppressed near P. subcostata algal fronds on GYP agar plates. These results suggest that HOX plays a role in P. subcostata resistance to bacterial attack by mediating H2O2 production in the marine environment.

The µ Subunit of Arabidopsis Adaptor Protein-2 Is Involved in Effector-Triggered Immunity Mediated by Membrane-Localized Resistance Proteins.

Endocytosis has been suggested to be important in the cellular processes of plant immune responses. However, our understanding of its role during effector-triggered immunity (ETI) is still limited. We have previously shown that plant endocytosis, especially clathrin-coated vesicle formation at the plasma membrane, is mediated by the adaptor protein-2 (AP-2) complex and that loss of the µ subunit of AP-2 (AP2M) affects plant growth and floral organ development. Here, we report that AP2M is required for full-strength ETI mediated by the disease resistance (R) genes RPM1 and RPS2 in Arabidopsis. Reduced ETI was observed in an ap2m mutant plant, measured by growth of Pseudomonas syringae pv. tomato DC3000 strains carrying the corresponding effector genes avrRpm1 or avrRpt2 and by hypersensitive cell death response and defense gene expression triggered by these strains. In contrast, RPS4-mediated ETI and its associated immune responses were not affected by the ap2m mutation. While RPM1 and RPS2 are localized to the plasma membrane, RPS4 is localized to the cytoplasm and nucleus. Our results suggest that AP2M is involved in ETI mediated by plasma membrane-localized R proteins, possibly by mediating endocytosis of the immune receptor complex components from the plasma membrane.

Pectin Biosynthesis Is Critical for Cell Wall Integrity and Immunity in Arabidopsis thaliana.

Plant cell walls are important barriers against microbial pathogens. Cell walls of Arabidopsis thaliana leaves contain three major types of polysaccharides: cellulose, various hemicelluloses, and pectins. UDP-D-galacturonic acid, the key building block of pectins, is produced from the precursor UDP-D-glucuronic acid by the action of glucuronate 4-epimerases (GAEs). Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) repressed expression of GAE1 and GAE6 in Arabidopsis, and immunity to Pma ES4326 was compromised in gae6 and gae1 gae6 mutant plants. These plants had brittle leaves and cell walls of leaves had less galacturonic acid. Resistance to specific Botrytis cinerea isolates was also compromised in gae1 gae6 double mutant plants. Although oligogalacturonide (OG)-induced immune signaling was unaltered in gae1 gae6 mutant plants, immune signaling induced by a commercial pectinase, macerozyme, was reduced. Macerozyme treatment or infection with B. cinerea released less soluble uronic acid, likely reflecting fewer OGs, from gae1 gae6 cell walls than from wild-type Col-0. Although both OGs and macerozyme-induced immunity to B. cinerea in Col-0, only OGs also induced immunity in gae1 gae6. Pectin is thus an important contributor to plant immunity, and this is due at least in part to the induction of immune responses by soluble pectin, likely OGs, that are released during plant-pathogen interactions.

Putative Serine Protease Effectors of Clavibacter michiganensis Induce a Hypersensitive Response in the Apoplast of Nicotiana Species.

Clavibacter michiganensis subspp. michiganensis and sepedonicus cause diseases on solanaceous crops. The genomes of both subspecies encode members of the pat-1 family of putative serine proteases known to function in virulence on host plants and induction of hypersensitive responses (HR) on nonhosts. One gene of this family in C. michiganensis subsp. sepedonicus, chp-7, is required for triggering HR in Nicotiana tabacum. Here, further investigation revealed that mutation of the putative catalytic serine residue at position 232 to threonine abolished the HR induction activity of Chp-7, suggesting that enzymatic activity is required. Purified Chp-7 triggered an HR in N. tabacum leaves in the absence of the pathogen, indicating Chp-7 itself is the HR elicitor from C. michiganensis subsp. sepedonicus. Ectopic expression of chp-7 constructs in N. tabacum leaves revealed that Chp-7 targeted to the apoplast triggered an HR while cytoplasmic Chp-7 did not, indicating that Chp-7 induces the HR in the apoplast of N. tabacum leaves. Chp-7 also induced HR in N. sylvestris, a progenitor of N. tabacum, but not in other Nicotiana species tested. ChpG, a related protein from C. michiganensis subsp. michiganensis, also triggered HR in N. tabacum and N. sylvestris. Unlike Chp-7, ChpG triggered HR in N. clevelandii and N. glutinosa.

Vacuolar processing enzyme in plant programmed cell death.

Vacuolar processing enzyme (VPE) is a cysteine proteinase originally identified as the proteinase responsible for the maturation and activation of vacuolar proteins in plants, and it is known to be an ortholog of animal asparaginyl endopeptidase (AEP/VPE/legumain). VPE has been shown to exhibit enzymatic properties similar to that of caspase 1, which is a cysteine protease that mediates the programmed cell death (PCD) pathway in animals. Although there is limited sequence identity between VPE and caspase 1, their predicted three-dimensional structures revealed that the essential amino-acid residues for these enzymes form similar pockets for the substrate peptide YVAD. In contrast to the cytosolic localization of caspases, VPE is localized in vacuoles. VPE provokes vacuolar rupture, initiating the proteolytic cascade leading to PCD in the plant immune response. It has become apparent that the VPE-dependent PCD pathway is involved not only in the immune response, but also in the responses to a variety of stress inducers and in the development of various tissues. This review summarizes the current knowledge on the contribution of VPE to plant PCD and its role in vacuole-mediated cell death, and it also compares VPE with the animal cell death executor caspase 1.

The receptor-like cytoplasmic kinase PCRK1 contributes to pattern-triggered immunity against Pseudomonas syringae in Arabidopsis thaliana.

In this paper we describe PATTERN-TRIGGERED IMMUNITY (PTI) COMPROMISED RECEPTOR-LIKE CYTOPLASMIC KINASE 1 (PCRK1) of Arabidopsis thaliana, an RLCK that is important for defense against the pathogen Pseudomonas syringae pv. maculicola ES4326 (Pma ES4326). We examined defense responses such as bacterial growth, production of reactive oxygen species (ROS) and callose deposition in pcrk1 mutant plants to determine the role of PCRK1 during pathogen infection. Expression of PCRK1 was induced following pathogen infection. Pathogen growth was significantly higher in pcrk1 mutant lines than in wild-type Col-0. Mutant pcrk1 plants showed reduced pattern-triggered immunity (PTI) against Pma ES4326 after pretreatment with peptides derived from flagellin (flg22), elongation factor-Tu (elf18), or an endogenous protein (pep1). Deposition of callose was reduced in pcrk1 plants, indicating a role of PCRK1 in activation of early immune responses. A PCRK1 transgene containing a mutation in a conserved lysine residue important for phosphorylation activity of kinases (K118E) failed to complement a pcrk1 mutant for the Pma ES4326 growth phenotype. Our study shows that PCRK1 plays an important role during PTI and that a conserved lysine residue in the putative kinase domain is important for PCRK1 function.

BEACH-domain proteins act together in a cascade to mediate vacuolar protein trafficking and disease resistance in Arabidopsis.

Membrane trafficking to the protein storage vacuole (PSV) is a specialized process in seed plants. However, this trafficking mechanism to PSV is poorly understood. Here, we show that three types of Beige and Chediak-Higashi (BEACH)-domain proteins contribute to both vacuolar protein transport and effector-triggered immunity (ETI). We screened a green fluorescent seed (GFS) library of Arabidopsis mutants with defects in vesicle trafficking and isolated two allelic mutants gfs3 and gfs12 with a defect in seed protein transport to PSV. The gene responsible for the mutant phenotype was found to encode a putative protein belonging to group D of BEACH-domain proteins, which possess kinase domains. Disruption of other BEACH-encoding loci in the gfs12 mutant showed that BEACH homologs acted in a cascading manner for PSV trafficking. The epistatic genetic interactions observed among BEACH homologs were also found in the ETI responses of the gfs12 and gfs12 bchb-1 mutants, which showed elevated avirulent bacterial growth. The GFS12 kinase domain interacted specifically with the pleckstrin homology domain of BchC1. These results suggest that a cascade of multiple BEACH-domain proteins contributes to vacuolar protein transport and plant defense.

Identification and dynamics of Arabidopsis adaptor protein-2 complex and its involvement in floral organ development.

The adaptor protein-2 (AP-2) complex is a heterotetramer involved in clathrin-mediated endocytosis of cargo proteins from the plasma membrane in animal cells. The homologous genes of AP-2 subunits are present in the genomes of plants; however, their identities and roles in endocytic pathways are not clearly defined in plants. Here, we reveal the molecular composition of the AP-2 complex of Arabidopsis thaliana and its dynamics on the plasma membrane. We identified all of the α-, ß-, σ-, and µ-subunits of the AP-2 complex and detected a weak interaction of the AP-2 complex with clathrin heavy chain. The µ-subunit protein fused to green fluorescent protein (AP2M-GFP) was localized to the plasma membrane and to the cytoplasm. Live-cell imaging using a variable-angle epifluorescence microscope revealed that AP2M-GFP transiently forms punctate structures on the plasma membrane. Homozygous ap2m mutant plants exhibited abnormal floral structures, including reduced stamen elongation and delayed anther dehiscence, which led to a failure of pollination and a subsequent reduction of fertility. Our study provides a molecular basis for understanding AP-2-dependent endocytic pathways in plants and their roles in floral organ development and plant reproduction.

Luminescent proteins for high-speed single-cell and whole-body imaging.

The use of fluorescent proteins has revolutionized our understanding of biological processes. However, the requirement for external illumination precludes their universal application to the study of biological processes in all tissues. Although light can be created by chemiluminescence, light emission from existing chemiluminescent probes is too weak to use this imaging modality in situations when fluorescence cannot be used. Here we report the development of the brightest luminescent protein to date, Nano-lantern, which is a chimera of enhanced Renilla luciferase and Venus, a fluorescent protein with high bioluminescence resonance energy transfer efficiency. Nano-lantern allows real-time imaging of intracellular structures in living cells with spatial resolution equivalent to fluorescence and sensitive tumour detection in freely moving unshaved mice. We also create functional indicators based on Nano-lantern that can image Ca(2+), cyclic adenosine monophosphate and adenosine 5'-triphosphate dynamics in environments where the use of fluorescent indicators is not feasible. These luminescent proteins allow visualization of biological phenomena at previously unseen single-cell, organ and whole-body level in animals and plants.

Changes in cytosolic ATP levels and intracellular morphology during bacteria-induced hypersensitive cell death as revealed by real-time fluorescence microscopy imaging.

Hypersensitive cell death is known to involve dynamic remodeling of intracellular structures that uses energy released during ATP hydrolysis. However, the relationship between intracellular structural changes and ATP levels during hypersensitive cell death remains unclear. Here, to visualize ATP dynamics directly in real time in individual living plant cells, we applied a genetically encoded Förster resonance energy transfer (FRET)-based fluorescent ATP indicator, ATeam1.03-nD/nA, for plant cells. Intracellular ATP levels increased approximately 3 h after inoculation with the avirulent strain DC3000/avrRpm1 of Pseudomonas syringae pv. tomato (Pst), which was accompanied by the simultaneous disappearance of transvacuolar strands and appearance of bulb-like structures within the vacuolar lumen. Approximately 5 h after bacterial inoculation, the bulb-like structures disappeared and ATP levels drastically decreased. After another 2 h, the large central vacuole was disrupted. In contrast, no apparent changes in intracellular ATP levels were observed in the leaves inoculated with the virulent strain Pst DC3000. The Pst DC3000/avrRpm1-induced hypersensitive cell death was strongly suppressed by inhibiting ATP synthesis after oligomycin A application within 4 h after bacterial inoculation. When the inhibitor was applied 7 h after bacterial inoculation, cell death was unaffected. These observations show that changes in intracellular ATP levels correlate with intracellular morphological changes during hypersensitive cell death, and that ATP is required just before vacuolar rupture in response to bacterial infection.

Auto-luminescent genetically-encoded ratiometric indicator for real-time Ca2+ imaging at the single cell level.

BACKGROUND: Efficient bioluminescence resonance energy transfer (BRET) from a bioluminescent protein to a fluorescent protein with high fluorescent quantum yield has been utilized to enhance luminescence intensity, allowing single-cell imaging in near real time without external light illumination. METHODOLOGY/PRINCIPAL FINDINGS: We applied BRET to develop an autoluminescent Ca(2+) indicator, BRAC, which is composed of Ca(2+)-binding protein, calmodulin, and its target peptide, M13, sandwiched between a yellow fluorescent protein variant, Venus, and an enhanced Renilla luciferase, RLuc8. Adjusting the relative dipole orientation of the luminescent protein's chromophores improved the dynamic range of BRET signal change in BRAC up to 60%, which is the largest dynamic range among BRET-based indicators reported so far. Using BRAC, we demonstrated successful visualization of Ca(2+) dynamics at the single-cell level with temporal resolution at 1 Hz. Moreover, BRAC signals were acquired by ratiometric imaging capable of canceling out Ca(2+)-independent signal drifts due to change in cell shape, focus shift, etc. CONCLUSIONS/SIGNIFICANCE: The brightness and large dynamic range of BRAC should facilitate high-sensitive Ca(2+) imaging not only in single live cells but also in small living subjects.