Search

With an efficient atom economy, low activation energy, and valuable applications for fuel cells and hydrogen storage, formic acid (FA) is a useful fuel product to convert CO 2 and reduce emissions. Although metal catalysts are typically used for this conversion, unwanted side reactions remain a concern, particularly when products are attempted to be recovered long-term. In this study, an enzymatic catalyst is used to enable the selective conversion of CO 2 to FA, as a formate ion. A dual-cell flow reactor system is used to first reduce a charge mediator electrochemically (reduction cell), which then activates a catalyst to selectively convert CO 2 to formate (production cell). This approach minimizes enzyme degradation by avoiding direct contact with increased voltages and improves the quantity of formate produced. The system produced 25 mM of formate and reached over 50% Coulombic efficiency. The larger volume of this dual-cell system increases the quantity of formate produced beyond that of a batch cell. Additional design configurations are employed, including a pH control pump to maintain catalyst activity and a packed bed reactor to improve contact of the charge carrier with the catalyst. Both configurations retained higher production and efficiency long-term (∼168 h). The results highlight the challenges of developing a system where many parameters play a role in optimizing performance. Nevertheless, the ability of the system to produce formate from CO 2 demonstrates the potential to improve upon this configuration for a variety of electrochemical CO 2 conversion applications., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Formate dehydrogenase (FDH) is critical for the conversion between formate and carbon dioxide. Despite its importance, the structural complexity of FDH and difficulties in the production of the enzyme have made elucidating its unique physicochemical properties challenging. Here, we purified recombinant Methylobacterium extorquens AM1 FDH (MeFDH1) and used cryo-electron microscopy to determine its structure. We resolved a heterodimeric MeFDH1 structure at a resolution of 2.8 Å, showing a noncanonical active site and a well-embedded Fe-S redox chain relay. In particular, the tungsten bis-molybdopterin guanine dinucleotide active site showed an open configuration with a flexible C-terminal cap domain, suggesting structural and dynamic heterogeneity in the enzyme., (© 2024. The Author(s).)

Carbon monoxide dehydrogenase (CODH), formate dehydrogenase (FDH), hydrogenase (H2ase), and nitrogenase (N2ase) are crucial enzymatic catalysts that facilitate the conversion of industrially significant gases such as CO, CO 2 , H 2 , and N 2 . The tunnels in the gas-converting enzymes serve as conduits for these low molecular weight gases to access deeply buried catalytic sites. The identification of the substrate tunnels is imperative for comprehending the substrate selectivity mechanism underlying these gas-converting enzymes. This knowledge also holds substantial value for industrial applications, particularly in addressing the challenges associated with separation and utilization of byproduct gases. In this comprehensive review, we delve into the emerging field of tunnel engineering, presenting a range of approaches and analyses. Additionally, we propose methodologies for the systematic design of enzymes, with the ultimate goal of advancing protein engineering strategies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Leucine-rich repeat receptor kinases (LRR-RKs) play a pivotal role in diverse aspects of growth, development, and immunity in plants by sensing extracellular signals. Typically, LRR-RKs are activated through the ligand-induced interaction with a SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) coreceptor, triggering downstream signaling. ROOT MERISTEM GROWTH FACTOR1 (RGF1) INSENSITIVEs (RGIs) LRR-RLK receptors promote primary root meristem activity while inhibiting lateral root (LR) development in response to RGF peptide. In this study, we employed rapamycin-induced dimerization (RiD) and BAK1-INTERACTING RECEPTOR-LIKE KINASE3 (BIR3) chimera approaches to explore the gain-of-function of RGI1, RGI4, and RGI5. Rapamycin induced the association of cytosolic kinase domains (CKDs) of RGI1 and the BAK1 coreceptor, activating both mitogen-activated protein kinase 3 (MPK3) and MPK6. Rapamycin significantly inhibited LR formation in RiD-RGI1/RGI4/RGI5-BAK1 plants. Using transgenic Arabidopsis expressing RGI1CKD fused to the BIR3-LRR chimera under estradiol control, we observed a substantial reduction in LR density upon β-estradiol treatment. Additionally, we identified a decrease in root gravitropism in BIR3 chimera plants. In contrast, RiD-RGI/BAK1 plants did not exhibit defects in root gravitropism, implying the importance of combinatorial interactions between RGIs and SERK coreceptors in the inhibition of root gravitropism. Constitutive activation of RGIs with BAK1 in RiD-RGI/BAK1 plants by rapamycin treatment resulted in the inhibition of primary root growth, resembling the inhibitory effects observed with high concentrations of phytohormones on primary root elongation. Our findings highlight that the interactions between CKDs of RGIs and BAK1, constitutively induced by rapamycin or BIR3 chimera, efficiently control LR development., (© 2024 Scandinavian Plant Physiology Society.)

ROOT MERISTEM GROWTH FACTOR1 (RGF1) and its receptors RGF1 INSENSITIVEs (RGIs), a group of leucine-rich repeat receptor kinases, promote primary root meristem activity via a mitogen-activated protein kinase (MPK) signaling cascade and control root gravitropism in Arabidopsis. Genetic analyses and in vitro binding assays have indicated that among five RGIs identified in Arabidopsis, RGI1, RGI2, and RGI3 recognize RGF1 peptides. However, it remains unclear whether the RGF1 peptide is redundantly recognized by these RGIs or mainly by a single RGI in the regulation of primary root meristem activity. In the present study, we analyzed root meristem growth of the rgi1 , rgi2 , and rgi3 single mutants in response to RGF1 treatment and observed a significantly decreased sensitivity in meristem growth of rgi1 and complete insensitivity in rgi1 rgi2 rgi3 triple mutant compared with the wild type but not in the rgi1 and rgi2 single mutants. We also observed that both root gravitropism and meristem growth in the BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE 1 ( bak1 ) single mutant were insensitive to RGF1 peptide treatment, whereas other serk mutants, such as serk1 , serk2 , and serk4 , were fully sensitive to RGF1 peptide like the wild type. These mutant analyses suggest that RGI1-BAK1 pair acts as the main receptor-coreceptor pair for regulating primary root gravitropism and meristem activity in response to RGF1 peptide in Arabidopsis.

It is challenging to capture carbon dioxide (CO 2 ), a major greenhouse gas in the atmosphere, due to its high chemical stability. One potential practical solution to eliminate CO 2 is to convert CO 2 into formate using hydrogen (H 2 ) (CO 2 hydrogenation), which can be accomplished with inexpensive hydrogen from sustainable sources. While industrial flue gas could provide an adequate source of hydrogen, a suitable catalyst is needed that can tolerate other gas components, such as carbon monoxide (CO) and oxygen (O 2 ), potential inhibitors. Our proposed CO 2 hydrogenation system uses the hydrogenase derived from Ralstonia eutropha H16 (ReSH) and formate dehydrogenase derived from Methylobacterium extorquens AM1 (MeFDH1). Both enzymes are tolerant to CO and O 2 , which are typical inhibitors of metalloenzymes found in flue gas. We have successfully demonstrated that combining ReSH- and MeFDH1-immobilized resins can convert H 2 and CO 2 in real flue gas to formate via a nicotinamide adenine dinucleotide-dependent cascade reaction. We anticipated that this enzyme system would enable the utilization of diverse H 2 and CO 2 sources, including waste gases, biomass, and gasified plastics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Cha, Lee, Jeon, Kim and Kwon.)

Methylorubrum extorquens AM1 has the potential to consume C1 feedstock to produce a wide range of biomaterials, from bioplastic to pharmaceutical. However, the synthetic biology tools for engineering M. extorquens AM1 need to be employed for precise control of recombinant enzyme expression. In this study, we presented an approach to improve the expression level of formate dehydrogenase 1 from M. extorquens AM1 (MeFDH1) using an efficient terminator and 5'-untranslated region (5'-UTR) design for enhanced carbon dioxide (CO 2 ) conversion activity of whole-cell biocatalyst. The rrnB terminator significantly increased mRNA levels of MeFDH1 alpha and beta subunits by 8.2-fold and 11-fold, respectively, compared to the T7 terminator. Moreover, enzyme production was 1.6-fold higher with 2.1 mg/wet cell weight (WCW) using rrnB terminator. Homologous 5'-untranslated regions (5'-UTR) determined based on proteomics data and UTR designer also influenced the expression level of MeFDH1. The 5'-UTR of the formaldehyde activating enzyme (fae) was the strongest, with 2.5-fold higher expression than that of the control sequence (T7g-10L). Furthermore, the electrochemical reaction of recombinant strains as whole-cell biocatalysts was investigated for their applicability to CO 2 conversion, showing enhanced formate productivity. The recombinant strain containing the 5'-UTR sequence of fae exhibited formate productivity of 5.0 mM/h, 2.3-fold higher than that of the control strain (T7). Overall, this study suggested practical applications for CO 2 conversion into bioavailable formate and provided valuable insights for recombinant expression systems in methylotrophic strains., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

ROOT MERISTEM GROWTH FACTOR1 (RGF1) and its receptors RGF1 INSENSITIVEs (RGIs) regulate primary root meristem activity via a mitogen-activated protein kinase (MPK) signaling cascade in Arabidopsis. However, it is unknown how RGF1 regulates lateral root (LR) development. Here, we show that the RGF1-RGI1 peptide-receptor pair negatively regulates LR development via activation of PUCHI encoding AP2/EREBP. Exogenous RGF1 peptides inhibited LR development of the wild type. However, the rgi1 mutants were partially or fully insensitive to RGF1 during LR development, whereas four other rgi single mutants, namely rgi2, rgi3, rgi4, and rgi5, were sensitive to RGF1 in inhibiting LR formation. Consistent with this, the red fluorescent protein (RFP) signals driven by the RGF1 promoter were detected at stage I and the following stages, overlapping with RGI1 expression. PUCHI expression was significantly up-regulated by RGF1 but completely inhibited in rgi1. LR development of puchi1-1 was insensitive to RGF1. PUCHI expression driven by the RGI1 promoter reduced LR density in both the wild type and rgi1,2,3. Further, mpk6, but not mpk3, displayed significantly down-regulated PUCHI expression and insensitive LR development in response to RGF1. Collectively, these results suggest that the RGF1-RGI1 module negatively regulates LR development by activating PUCHI expression via MPK6., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Membrane-localized leucine-rich repeat receptor kinases (LRR-RKs) sense diverse extracellular signals, and coordinate and specify cellular functions in plants. However, functional understanding and identification of the cellular signaling of most LRR-RKs remain a major challenge owing to their genetic redundancy, the lack of ligand information, and subtle phenotypes of LRR-RK overexpression. Here, we report an engineered rapamycin-inducible dimerization (RiD) receptor system that triggers a receptor-specific LRR-RK signaling independent of their cognate ligands or endogenous receptors. Using the RiD-receptors, we demonstrated that the rapamycin-mediated association of chimeric cytosolic kinase domains from the BRI1/BAK1 receptor/co-receptor, but not the BRI1/BRI1 or BAK1/BAK1 homodimer, is sufficient to activate downstream brassinosteroid signaling and physiological responses. Furthermore, we showed that the engineered RiD-FLS2/BAK1 could activate flagellin-22-mediated immune signaling and responses. Using the RiD system, we also identified the potential function of an unknown orphan receptor in immune signaling and revealed the differential activities of SERK co-receptors of LRR-RKs. Our results indicate that the RiD method can serve as a synthetic biology tool for precise temporal manipulation of LRR-RK signaling and for understanding LRR-RK biology., (Copyright © 2021 The Author. Published by Elsevier Inc. All rights reserved.)

Plant signaling peptides are involved in cell-cell communication networks and coordinate a wide range of plant growth and developmental processes. Signaling peptides generally bind to receptor-like kinases, inducing their dimerization with co-receptors for signaling activation to trigger cellular signaling and biological responses. Fertilization is an important life event in flowering plants, involving precise control of cell-cell communications between male and female tissues. Peptide-receptor-like kinase-mediated signaling plays an important role in male-female interactions for successful fertilization in flowering plants. Here, we describe the recent findings on the functions and signaling pathways of peptides and receptors involved in plant reproduction processes including pollen germination, pollen tube growth, pollen tube guidance to the embryo sac, and sperm cell reception in female tissues., Competing Interests: Declaration of Interests No interests are declared., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

As sessile organisms, plants are exposed to constantly changing environments that are often stressful for their growth and development. To cope with these stresses, plants have evolved complex and sophisticated stress-responsive signaling pathways regulating the expression of transcription factors and biosynthesis of osmolytes that confer tolerance to plants. Signaling peptides acting like phytohormones control various aspects of plant growth and development via cell-cell communication networks. These peptides are typically recognized by membrane-embedded receptor-like kinases, inducing activation of cellular signaling to control plant growth and development. Recent studies have revealed that several signaling peptides play important roles in plant responses to abiotic stress. In this mini review, we provide recent findings on the roles and signaling pathways of peptides that are involved in coordinating plant responses to abiotic stresses, such as dehydration, high salinity, reactive oxygen species, and heat. We also discuss recent developments in signaling peptides that play a role in plant adaptation responses to nutrient deficiency stress, focusing on nitrogen and phosphate deficiency responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kim, Jeon and Kim.)

Roots provide the plant with water and nutrients and anchor it in a substrate. Root development is controlled by plant hormones and various sets of transcription factors. Recently, various small peptides and their cognate receptors have been identified as controlling root development. Small peptides bind to membrane-localized receptor-like kinases, inducing their dimerization with co-receptor proteins for signaling activation and giving rise to cellular signaling outputs. Small peptides function as local and long-distance signaling molecules involved in cell-to-cell communication networks, coordinating root development. In this review, we survey recent advances in the peptide ligand-mediated signaling pathways involved in the control of root development in Arabidopsis. We describe the interconnection between peptide signaling and conventional phytohormone signaling. Additionally, we discuss the diversity of identified peptide-receptor interactions during plant root development., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In 2017, for the first time in Asia, we reported the isolation of variants of Avibacterium paragallinarum with atypical NAD dependency. The present study was conducted to characterize the genotypes of 24 isolates of Av. paragallinarum in Korea, including the four variants reported previously. Most of the typical isolates (19/20) showed a unique ERIC-PCR pattern with no ERIC-PCR patterns in common between the typical isolates and the variants. Furthermore, the variants shared no ERIC-PCR patterns among themselves. All the typical NAD-dependent isolates belonged to the same phylogenetic group based on both 16S rRNA and hagA gene sequences. The four variants were placed in several groups distinct from the typical isolates. In the 16S rRNA phylogenetic analysis, two of the variants were not closely aligned to any other Av. paragallinarum, isolate although they were clearly members of the genus Avibacterium . The other variants were clustered together with NAD atypical isolates from geographically diverse global locations. Compared with the Modesto reference strain AY498870, all the variants lacked a TTTTT stretch at positions 182-186 in the 16S rRNA gene and the same deletion was shown in most of the reported variants. The typical isolates and variants shared 97.3-98.2% and 95.2-97.2% nucleotide sequence similarity, for 16S rRNA and hagA , respectively. In addition, the similarities among variants were within 98.3-100% and 96.5-98.4% for the two genes, respectively. Our results indicate that the Av. paragallinarum variants with altered NAD growth requirements were genetically different and highly divergent from the typical NAD-dependent isolates. RESEARCH HIGHLIGHTS NAD variant Korean Av. paragallinarum isolates show genetic diversity, whereas typical Korean Av. paragallinarum isolates do not.The Korean variants were not closely aligned to all other Av. paragallinarum in the 16S rRNA phylogeny.NAD atypical isolates from geographically diverse global locations clustered together.Almost all variants, including all Korean variants of Av. paragallinarum , lack a specific fragment of the 16S rRNA gene.

Cytosolic calcium concentration ([Ca 2+ ] 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; [Ca 2+ ] 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 [Ca 2+ ] cyt oscillations in response to Ca o , initiating only a single [Ca 2+ ] cyt spike. Experimentally imposed [Ca 2+ ] 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 Ca 2+ -induced Ca 2+ release that is likely dependent on Gβγ interaction with PLCs leading to InsP3 production., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

The conversion of carbon dioxide to formate is a fundamental step for building C1 chemical platforms. Methylobacterium extorquens AM1 was reported to show remarkable activity converting carbon dioxide into formate. Formate dehydrogenase 1 from M. extorquens AM1 (MeFDH1) was verified as the key responsible enzyme for the conversion of carbon dioxide to formate in this study. Using a 2% methanol concentration for induction, microbial harboring the recombinant MeFDH1 expressing plasmid produced the highest concentration of formate (26.6 mM within 21 hours) in electrochemical reactor. 60 μM of sodium tungstate in the culture medium was optimal for the expression of recombinant MeFDH1 and production of formate (25.7 mM within 21 hours). The recombinant MeFDH1 expressing cells showed maximum formate productivity of 2.53 mM/g-wet cell/hr, which was 2.5 times greater than that of wild type. Thus, M. extorquens AM1 was successfully engineered by expressing MeFDH1 as recombinant enzyme to elevate the production of formate from CO 2 after elucidating key responsible enzyme for the conversion of CO 2 to formate.

The LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) gene family encode plant-specific transcription factors that regulate various aspects of plant growth and development. Arabidopsis genome has 42 LBD genes. Several LBD genes, such as LBD16, -18, -29, and -33, have been shown to function in lateral root (LR) development via auxin signaling. Although abscisic acid (ABA) is a well-known antistress plant hormone regulating various plant developmental processes, it also plays a role in LR growth regulation. Our recent study showed that LBD14 expression is downregulated by ABA during the entire steps of LR development. The RNAi-induced downregulation and overexpression of LBD14 indicated that LBD14 promotes LR formation. LBD14RNAi enhanced the ABA-induced suppression of LR density compared with the wild type, suggesting that LBD14 is involved in the ABA-mediated control of LR formation. Our study provides an insight into the signaling mechanism of developmental plasticity whereby ABA controls LR branching via LBD14 downregulation under abiotic stress conditions.

Stomata, microscopic pores in leaf surfaces through which water loss and carbon dioxide uptake occur, are closed in response to drought by the phytohormone abscisic acid (ABA). This process is vital for drought tolerance and has been the topic of extensive experimental investigation in the last decades. Although a core signaling chain has been elucidated consisting of ABA binding to receptors, which alleviates negative regulation by protein phosphatases 2C (PP2Cs) of the protein kinase OPEN STOMATA 1 (OST1) and ultimately results in activation of anion channels, osmotic water loss, and stomatal closure, over 70 additional components have been identified, yet their relationships with each other and the core components are poorly elucidated. We integrated and processed hundreds of disparate observations regarding ABA signal transduction responses underlying stomatal closure into a network of 84 nodes and 156 edges and, as a result, established those relationships, including identification of a 36-node, strongly connected (feedback-rich) component as well as its in- and out-components. The network's domination by a feedback-rich component may reflect a general feature of rapid signaling events. We developed a discrete dynamic model of this network and elucidated the effects of ABA plus knockout or constitutive activity of 79 nodes on both the outcome of the system (closure) and the status of all internal nodes. The model, with more than 1024 system states, is far from fully determined by the available data, yet model results agree with existing experiments in 82 cases and disagree in only 17 cases, a validation rate of 75%. Our results reveal nodes that could be engineered to impact stomatal closure in a controlled fashion and also provide over 140 novel predictions for which experimental data are currently lacking. Noting the paucity of wet-bench data regarding combinatorial effects of ABA and internal node activation, we experimentally confirmed several predictions of the model with regard to reactive oxygen species, cytosolic Ca2+ (Ca2+c), and heterotrimeric G-protein signaling. We analyzed dynamics-determining positive and negative feedback loops, thereby elucidating the attractor (dynamic behavior) repertoire of the system and the groups of nodes that determine each attractor. Based on this analysis, we predict the likely presence of a previously unrecognized feedback mechanism dependent on Ca2+c. This mechanism would provide model agreement with 10 additional experimental observations, for a validation rate of 85%. Our research underscores the importance of feedback regulation in generating robust and adaptable biological responses. The high validation rate of our model illustrates the advantages of discrete dynamic modeling for complex, nonlinear systems common in biology.

Twenty field isolates of Avibacterium paragallinarum were obtained from chickens in South Korea during 2011-2015. The isolates were identified by a HPG-2 PCR assay specific for A. paragallinarum and by biochemical tests. Growth requirements, Page serovars, carbohydrate fermentation patterns, and antimicrobial susceptibility were also examined. Most isolates (16/20) showed the typical requirement for nicotinamide adenine dinucleotide (NAD) and an enriched CO 2 atmosphere for growth. One isolate needed increased levels of NAD and serum for good growth. Three isolates showed NAD-independent growth on blood agar under aerobic conditions. In terms of carbohydrate fermentation patterns, three biochemical biovars were recognized; these varied with respect to acid production from maltose and D-xylose. The 16 typical NAD-dependent isolates were serovar A while the variants, both NAD-independent isolates and the isolate with increased NAD dependency were non-typeable. All isolates were sensitive to amoxicillin-clavulanic acid, ceftiofur, gentamicin, and spectinomycin. High rates of resistance, including intermediate resistance, to lincomycin (100%), cloxacillin (75%), and erythromycin (70%) were observed. The four variant strains (the three NAD-independent isolates and the isolate showing unusual growth requirements) were more resistant to antibiotics than the typical NAD-dependent strains. The finding of NAD-independent forms of A. paragallinarum extends the known distribution of this form, previously only reported in South Africa, Mexico and Peru. There is clearly a need for increased caution in the diagnosis and, possibly, the control of infectious coryza., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Salmonella enterica serovar Enteritidis is one of the most common serotypes implicated in Salmonella infections in both humans and poultry worldwide. It has been reported that human salmonellosis is mainly associated with the consumption of poultry products contaminated with serovar Enteritidis. The present study was to extensively analyze the public health risk of serovar Enteritidis isolates from chickens in Korea. A total of 127 chicken isolates were collected from clinical cases, on-farm feces, and chicken meat between 1998 and 2012 and 20 human clinical isolates were obtained from patients with diarrhea between 2000 and 2006 in Korea. To characterize the isolates from chickens and humans, we compared the pulsed-field gel electrophoresis (PFGE) patterns and multilocus variable-number tandem-repeat analysis (MLVA) profiles of the isolates. We further characterized representative isolates of different genotypes using a DNA microarray. PFGE revealed 28 patterns and MLVA identified 16 allelic profiles. The DNA microarray showed high genetic variability in plasmid regions and other fimbrial subunits of the isolates although the virulence gene contents of isolates from the same source and/or of the same genotype were unrelated. PFGE and MLVA showed that major genotypes were present in both human and chicken isolates. This result suggests that chickens in Korea pose a significant risk to public health as a source of serovar Enteritidis as has been noted in other countries., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Ascorbic acid (AsA) maintains redox homeostasis by scavenging reactive oxygen species from prokaryotes to eukaryotes, especially plants. The enzyme monodehydroascorbate reductase (MDHAR) regenerates AsA by catalysing the reduction of monodehydroascorbate, using NADH or NADPH as an electron donor. The detailed recycling mechanism of MDHAR remains unclear due to lack of structural information. Here, we present the crystal structures of MDHAR in the presence of cofactors, nicotinamide adenine dinucleotide (NAD + ) and nicotinamide adenine dinucleotide phosphate (NADP + ), and complexed with AsA as well as its analogue, isoascorbic acid (ISD). The overall structure of MDHAR is similar to other iron-sulphur protein reductases, except for a unique long loop of 63-80 residues, which seems to be essential in forming the active site pocket. From the structural analysis and structure-guided point mutations, we found that the Arg320 residue plays a major substrate binding role, and the Tyr349 residue mediates electron transfer from NAD(P)H to bound substrate via FAD. The enzymatic activity of MDHAR favours NADH as an electron donor over NADPH. Our results show, for the first time, structural insights into this preference. The MDHAR-ISD complex structure revealed an alternative binding conformation of ISD, compared with the MDHAR-AsA complex. This implies a broad substrate (antioxidant) specificity and resulting greater protective ability of MDHAR.

The cyanobacterial aldehyde deformylating oxygenase (cADO) is a key enzyme that catalyzes the unusual deformylation of aliphatic aldehydes for alkane biosynthesis and can be applied to the production of biofuel in vitro and in vivo. In this study, we determined crystal structures of two ADOs from Limnothrix sp. KNUA012 (LiADO) and Oscillatoria sp. KNUA011 (OsADO). The structures of LiADO and OsADO resembled those of typical cADOs, consisting of eight α-helices found in ferritin-like di-iron proteins. However, structural comparisons revealed that while the LiADO active site was vacant of iron and substrates, the OsADO active site was fully occupied, containing both a coordinated metal ion and substrate. Previous reports indicated that helix 5 is capable of adopting two distinct conformations depending upon the existence of bound iron. We observed that helix 5 of OsADO with an iron bound in the active site presented as a long helix, whereas helix 5 of LiADO, which lacked iron in the active site, presented two conformations (one long and two short helices), indicating that an equilibrium exists between the two states in solution. Furthermore, acquisition of a structure having a fully occupied active site is unique in the absence of higher iron concentrations as compared with other cADO structures, wherein low affinity for iron complicates the acquisition of crystal structures with bound iron. An in-depth analysis of the ADO apo-enzyme, the enzyme with substrate bound, and the enzyme with both iron and substrate bound provided novel insight into substrate-binding modes in the absence of a coordinated metal ion and suggested a separate two-step binding mechanism for substrate and iron co-factors. Moreover, our results provided a comprehensive structural basis for conformational changes induced by binding of the substrate and co-factor., (Copyright © 2016 Elsevier Inc. All rights reserved.)

1169 I. 1170 II. 1170 III. 1172 IV. 1176 V. 1181 VI. 1182 1183 References 1183 SUMMARY: Modern agriculture is facing multiple challenges including the necessity for a substantial increase in production to meet the needs of a burgeoning human population. Water shortage is a deleterious consequence of both population growth and climate change and is one of the most severe factors limiting global crop productivity. Brassica species, particularly canola varieties, are cultivated worldwide for edible oil, animal feed, and biodiesel, and suffer dramatic yield loss upon drought stress. The recent release of the Brassica napus genome supplies essential genetic information to facilitate identification of drought-related genes and provides new information for agricultural improvement in this species. Here we summarize current knowledge regarding drought responses of canola, including physiological and -omics effects of drought. We further discuss knowledge gained through translational biology based on discoveries in the closely related reference species Arabidopsis thaliana and through genetic strategies such as genome-wide association studies and analysis of natural variation. Knowledge of drought tolerance/resistance responses in canola together with research outcomes arising from new technologies and methodologies will inform novel strategies for improvement of drought tolerance and yield in this and other important crop species., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Dehydroascorbate reductase (DHAR) is a key enzyme involved in the recycling of ascorbate, which catalyses the glutathione (GSH)-dependent reduction of oxidized ascorbate (dehydroascorbate, DHA). As a result, DHAR regenerates a pool of reduced ascorbate and detoxifies reactive oxygen species (ROS). In previous experiments involving transgenic rice, we observed that overexpression of DHAR enhanced grain yield and biomass. Since the structure of DHAR is not available, the enzymatic mechanism is not well-understood and remains poorly characterized. To elucidate the molecular basis of DHAR catalysis, we determined the crystal structures of DHAR from Oryza sativa L. japonica (OsDHAR) in the native, ascorbate-bound, and GSH-bound forms and refined their resolutions to 1.9, 1.7, and 1.7 Å, respectively. These complex structures provide the first information regarding the location of the ascorbate and GSH binding sites and their interacting residues. The location of the ascorbate-binding site overlaps with the GSH-binding site, suggesting a ping-pong kinetic mechanism for electron transfer at the common Cys20 active site. Our structural information and mutagenesis data provide useful insights into the reaction mechanism of OsDHAR against ROS-induced oxidative stress in rice.

Bioassays are commonly used to study stomatal phenotypes. There are multiple options in the choice of plant materials and species used for observation of stomatal and guard cell responses in vivo. Here, detailed procedures for bioassays of stomatal responses to abscisic acid (ABA) in Arabidopsis thaliana are described, including ABA promotion of stomatal closure, ABA inhibition of stomatal opening, and ABA promotion of reaction oxygen species (ROS) production in guard cells. We also include an example of a stomatal bioassay for the guard cell CO2 response using guard cell-enriched epidermal peels from Brassica napus. Highly pure preparations of guard cell protoplasts can be produced, which are also suitable for studies on guard cell signaling, as well as for studies on guard cell ion transport. Small-scale and large-scale guard cell protoplast preparations are commonly used for electrophysiological and -omics studies, respectively. We provide a procedure for small-scale guard cell protoplasting from A. thaliana. Additionally, a general protocol for large-scale preparation of guard cell protoplasts, with specifications for three different species, A. thaliana, B. napus, and Vicia faba is also provided.

Stomata are the tiny valves on the plant surface that mediate gas exchange between the plant and its environment. Stomatal opening needs to be tightly regulated to facilitate CO2 uptake and prevent excess water loss. Plant Rho-type (ROP) GTPase 2 (ROP2) is a molecular component of the system that negatively regulates light-induced stomatal opening. Previously, ROP-interactive Cdc42- and Rac-interactive binding motif-containing protein 7 (RIC7) was suggested to function downstream of ROP2. However, the underlying molecular mechanism remains unknown. To understand the mechanism by which RIC7 regulates light-induced stomatal opening, we analyzed the stomatal responses of ric7 mutant Arabidopsis plants and identified the target protein of RIC7 using a yeast two-hybrid screen. Light-induced stomatal opening was promoted by ric7 knockout, whereas it was inhibited by RIC7 overexpression, indicating that RIC7 negatively regulates stomatal opening in Arabidopsis. RIC7 interacted with exocyst subunit Exo70 family protein B1 (Exo70B1), a component of the vesicle trafficking machinery. RIC7 and Exo70B1 localized to the plasma membrane region under light or constitutively active ROP2 conditions. The knockout mutant of Exo70B1 and ric7/exo70b1 exhibited retarded light-induced stomatal opening. Our results suggest that ROP2 and RIC7 suppress excess stomatal opening by inhibiting Exo70B1, which most likely participates in the vesicle trafficking required for light-induced stomatal opening., (© 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.)

Objectives: A chaperonin, PsyGroELS, from the Antarctic psychrophilic bacterium Psychrobacter sp. PAMC21119, was examined for its role in cold adaptation when expressed in a mesophilic Escherichia coli strain., Results: Growth of E. coli harboring PsyGroELS at 10 °C was increased compared to the control strain. A co-expression system using PsyGroELS was developed to increase productivity of the psychrophilic enzyme PsyEst9. PsyEst9 was cloned and expressed using three E. coli variants that co-expressed GroELS from PAMC21119, E. coli, or Oleispira antarctica RB8(T). Co-expression with PsyGroELS was more effective for the production of PsyEst9 compared tothe other chaperonins., Conclusion: PsyGroELS confers cold tolerance to E. coli, and shows potential as an effective co-expression system for the stable production of psychrophilic proteins.

Individual metabolites have been implicated in abscisic acid (ABA) signaling in guard cells, but a metabolite profile of this specialized cell type is lacking. We used liquid chromatography-multiple reaction monitoring mass spectrometry for targeted analysis of 85 signaling-related metabolites in Arabidopsis thaliana guard cell protoplasts over a time course of ABA treatment. The analysis utilized ∼ 350 million guard cell protoplasts from ∼ 30,000 plants of the Arabidopsis Columbia accession (Col) wild type and the heterotrimeric G-protein α subunit mutant, gpa1, which has ABA-hyposensitive stomata. These metabolomes revealed coordinated regulation of signaling metabolites in unrelated biochemical pathways. Metabolites clustered into different temporal modules in Col versus gpa1, with fewer metabolites showing ABA-altered profiles in gpa1. Ca(2+)-mobilizing agents sphingosine-1-phosphate and cyclic adenosine diphosphate ribose exhibited weaker ABA-stimulated increases in gpa1. Hormone metabolites were responsive to ABA, with generally greater responsiveness in Col than in gpa1. Most hormones also showed different ABA responses in guard cell versus mesophyll cell metabolomes. These findings suggest that ABA functions upstream to regulate other hormones, and are also consistent with G proteins modulating multiple hormonal signaling pathways. In particular, indole-3-acetic acid levels declined after ABA treatment in Col but not gpa1 guard cells. Consistent with this observation, the auxin antagonist α-(phenyl ethyl-2-one)-indole-3-acetic acid enhanced ABA-regulated stomatal movement and restored partial ABA sensitivity to gpa1.

Open Stomata 1 (OST1) (SnRK2.6 or SRK2E), a serine/threonine protein kinase, is a positive regulator in abscisic acid (ABA)-mediated stomatal response, but OST1-regulation of K(+) and Ca(2+) currents has not been studied directly in guard cells and it is unknown whether OST1 activity is limiting in ABA-mediated stomatal responses. We employed loss-of-function and gain-of-function approaches to study native ABA responses of Arabidopsis guard cells. We performed stomatal aperture bioassays, patch clamp analyses and reactive oxygen species (ROS) measurements. ABA inhibition of inward K(+) channels and light-induced stomatal opening are reduced in ost1 mutants while transgenic plants overexpressing OST1 show ABA hypersensitivity in these responses. ost1 mutants are insensitive to ABA-induced stomatal closure, regulation of slow anion currents, Ca(2+) -permeable channel activation and ROS production while OST1 overexpressing lines are hypersensitive for these responses, resulting in accelerated stomatal closure in response to ABA. Overexpression of OST1 in planta in the absence of ABA application does not affect basal apertures or ion currents. Moreover, we demonstrate the physical interaction of OST1 with the inward K(+) channel KAT1, the anion channel SLAC1, and the NADPH oxidases AtrbohD and AtrbohF. Our findings support OST1 as a critical limiting component in ABA regulation of stomatal apertures, ion channels and NADPH oxidases in Arabidopsis guard cells., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

R-lactide, a pivotal monomer for the production of poly (D-lactic acid) (PDLA) or stereocomplex poly (lactic acid) (PLA) was synthesized from alkyl (R)-lactate through a lipase-catalyzed reaction without racemization. From among several types of lipase, only lipase B from Candida antarctica (Novozym 435; CAL-B) was effective in the reaction that synthesized (R,R)-lactide. Enantiopure (R,R)-lactide, which consisted of over 99% enantiomeric excess, was synthesized from methyl (R)-lactate through CAL-B catalysis. Removal of the methanol by-product was critical to obtain a high level of lactide conversion. The (R,R)-lactide yield was 56% in a reaction containing 100 mg of Novozym 435, 10 mM methyl (R)-lactate and 1500 mg of molecular sieve 5A in methyl tert-butyl ether (MTBE). The important monomer (R,R)-lactide that is required for the production of the widely recognized bio-plastic PDLA and the PLA stereocomplex can be obtained using this novel synthetic method., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: The Salmonella Gallinarum (SG) lon/cpxR deletion mutant JOL916 was developed as a live vaccine candidate for fowl typhoid (FT), and a SG mutant secreting an Escherichia coli heat-labile enterotoxin B subunit (LTB), designated JOL1229, was recently constructed as an adjuvant strain for oral vaccination against FT. In this study, we evaluated the immunogenicity and protective properties of the SG mutant JOL916 and the LTB adjuvant strain JOL1229 in order to establish a prime and boost immunization strategy for each strain. In addition, we compared the increase in body weight, the immunogenicity, the egg production rates, and the bacteriological egg contamination of these strains with those of SG 9R, a widely used commercial vaccine., Results: Plasma IgG, intestinal secretory IgA (sIgA), and cell-mediated responses were significantly induced after a boost inoculation with a mixture of JOL916 and JOL1229, and significant reductions in the mortality of chickens challenged with a wild-type SG strain were observed in the immunized groups. There were no significant differences in increases in body weight, cell-mediated immune responses, or systemic IgG responses between our vaccine mixture and the SG 9R vaccine groups. However, there was a significant elevation in intestinal sIgA in chickens immunized with our mixture at 3 weeks post-prime-immunization and at 3 weeks post-boost-immunization, while sIgA levels in SG 9R-immunized chickens were not significantly elevated compared to the control. In addition, the SG strain was not detected in the eggs of chickens immunized with our mixture., Conclusion: Our results suggest that immunization with the LTB-adjuvant strain JOL1229 can significantly increase the immune response, and provide efficient protection against FT with no side effects on body weight, egg production, or egg contamination.

Escherichia coli strains were isolated from the feces of 130 diarrheic calves at different farms locations in Korea. The presence of the virulence genes, such as fanC, f41, f17a, eaeA, clpG, afa-8D, sta, stx1 and stx2, in each E. coli isolate was examined. Among the 314 isolates, 157 carried one or more of the virulence genes tested in this study. The most prevalent virulence gene was clpG (45.9%), although f17A (36.9%) and afa-8D (21.7%) were also frequently observed. The sta, stx1 and eaeA genes were detected in between approximately 13 and 17% of the isolates, and the fanC and fim41a genes were detected to a lesser extent. Collectively, our data indicated that diarrhea in calves in these locations can be ascribed to various virulence factors, and the pathogenesis may be more related to virulence genes such as, clpG, f17A, and afa-8D.

In our previous study, we constructed a vaccine candidate (JOL916) for fowl typhoid (FT). A live adjuvant Salmonella Gallinarum (SG) strain was generated in the present study to facilitate efficacious oral vaccination with this vaccine. The Escherichia coli eltB gene secreting heat-labile enterotoxin B subunit (LTB) was cloned into an Asd(+) plasmid pJHL65. This was transformed into a Δlon ΔcpxR Δasd SG strain and the resulting strain was designated JOL1229. Secretion of LTB from JOL1229 was confirmed with an immunoblot assay. To determine the optimal dose of the strain, 50 six-week-old female chickens were divided into five groups (Groups A-E, n=10 per group) and orally inoculated with various doses of JOL1229 and JOL916. In Group B (consisting of four parts JOL916 and one part JOL1229), significant cell-mediated immune responses, plasma IgG levels and intestinal secretary IgA levels were induced after inoculation with both strains. On challenge with the wild-type strain, significant reductions in mortality were observed in the group. In addition, after inoculation the LTB strain was not recovered in feces samples, and resulted in no, or very mild, gross lesions in the liver and spleen. Both CD4(+) and CD8(+) T-cells were significantly increased in peripheral blood samples from the chickens immunized with the LTB strain. Expression of the interleukin-6 (IL-6) gene in splenocytes was induced in the chickens immunized with the LTB strain. These results suggest that oral immunization with the LTB-adjuvant strain, in particular with the four parts JOL916 and one part JOL1229 mixture, increased the immune response and provided efficient protection against FT in chickens., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A new strategy to develop an effective vaccine is essential to control food-borne Salmonella enterica serovar Enteritidis infections. Bacterial ghosts (BGs), which are nonliving, Gram-negative bacterial cell envelopes, are generated by expulsion of the cytoplasmic contents from bacterial cells through controlled expression using the modified cI857/λ P(R)/gene E expression system. In the present study, the pJHL99 lysis plasmid carrying the mutated lambda pR37-cI857 repressor and PhiX174 lysis gene E was constructed and transformed in S. Enteritidis to produce a BG. Temperature induction of the lysis gene cassette at 42°C revealed quantitative killing of S. Enteritidis. The S. Enteritidis ghost was characterized using scanning and transmission electron microscopy to visualize the transmembrane tunnel structure and loss of cytoplasmic materials, respectively. The efficacy of the BG as a vaccine candidate was evaluated in a chicken model using 60 10-day-old chickens, which were divided into four groups (n = 15), A, B, C, and D. Group A was designated as the nonimmunized control group, whereas the birds in groups B, C, and D were immunized via the intramuscular, subcutaneous, and oral routes, respectively. The chickens from all immunized groups showed significant increases in plasma IgG and intestinal secretory IgA levels. The lymphocyte proliferation response and CD3(+) CD4(+) and CD3(+) CD8(+) T cell subpopulations were also significantly increased in all immunized groups. The data indicate that both humoral and cell-mediated immune responses are robustly stimulated. Based on an examination of the protection efficacy measured by observations of gross lesions in the organs and bacterial recovery, the candidate vaccine can provide efficient protection against virulent challenge.

ROP GTPases function as molecular switches in diverse cellular processes. Previously, we showed that ROP2 GTPase is activated upon light irradiation, and thereby negatively regulates light-induced stomatal opening. Here we studied the role of ROP2 during stomatal closure. The expression of a constitutively active form of ROP2 (CA-rop2) in Arabidopsis thaliana and Vicia faba resulted in slower and reduced stomatal closure in response to abscisic acid (ABA) and CO(2) . In contrast, the expression of a dominant-negative form of ROP2 (DN-rop2) and the knockout mutation of ROP2 (rop2 KO) promoted ABA-induced stomatal closure in Arabidopsis. As early as 10 min after ABA treatment, ROP2 was inactivated and translocated to the cytoplasm of the stomatal guard cells. To elucidate the mechanism by which active ROP2 suppresses stomatal closure, we monitored endocytotic membrane trafficking, which is regulated by Rho GTPases in animal cells. We found that the endocytosis of plasma membrane (PM), as tracked by FM4-64, was lower in CA-rop2-expressing guard cells than in those of wild-type plants, which suggests that active ROP2 suppresses the endocytotic internalization of PM, a process required for stomatal closure. Together, our results suggest that ROP2 is inactivated by ABA, and that this inactivation is required for the timely stomatal closure., (© 2011 Blackwell Publishing Ltd.)

A sensitive and rapid method based on liquid chromatography-triple-quadrupole tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI) has been developed and validated for the screening and confirmation of 44 exogenous anabolic steroids (29 parent steroids and 15 metabolites) in human urine. The method involves an enzymatic hydrolysis, liquid-liquid extraction, and detection by LC-MS/MS. A triple-quadrupole mass spectrometer was operated in positive ESI mode with selected reaction monitoring (SRM) mode for the screening and product ion scan mode for the confirmation. The protonated molecular ions were used as precursor ions for the SRM analysis and product ion scan. The intraday and interday precisions of the target analytes at concentrations of the minimum required performance levels for the screening were 2-14% and 2-15%, respectively. The limits of detection for the screening and confirmation method were 0.1-10 ng/mL and 0.2-10 ng/mL, respectively, for 44 steroids. This method was successfully applied to analysis of urine samples from suspected anabolic steroid abusers.

The enzymatic coproduction of biodiesel and glycerol carbonate by the transesterification of soybean oil was studied using lipase as catalyst in organic solvent. To produce biodiesel and glycerol carbonate simultaneously, experiments were designed sequentially. Enzyme screening, the molar ratio of dimethyl carbonate (DMC) to soybean oil, reaction temperature and solvent effects were investigated. The results of enzyme screening, at 100 g/L Novozym 435 (immobilized Candida antarctica lipase B), biodiesel and glycerol carbonate showed conversions of 58.7% and 50.7%, respectively. The optimal conditions were 60 °C, 100 g/L Novozym 435, 6.0:1 molar ratio with tert-butanol as solvent: 84.9% biodiesel and 92.0% glycerol carbonate production was achieved., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Heterotrimeric G proteins composed of Gα, Gβ, and Gγ subunits are important signalling agents in both animals and plants. In plants, G proteins modulate numerous responses, including abscisic acid (ABA) and pathogen-associated molecular pattern (PAMP) regulation of guard cell ion channels and stomatal apertures. Previous analyses of mutants deficient in the sole canonical Arabidopsis Gα subunit, GPA1, have shown that Gα-deficient guard cells are impaired in ABA inhibition of K(+) influx channels, and in pH-independent activation of anion efflux channels. ABA-induced Ca(2+) uptake through ROS-activated Ca(2+)-permeable channels in the plasma membrane is another key component of ABA signal transduction in guard cells, but the question of whether these channels are also dependent on Gα for their ABA response has not been evaluated previously. We used two independent Arabidopsis T-DNA null mutant lines, gpa1-3 and gpa1-4, to investigate this issue. We observed that gpa1 mutants are disrupted both in ABA-induced Ca(2+)-channel activation, and in production of reactive oxygen species (ROS) in response to ABA. However, in response to exogenous H(2)O(2) application, I(Ca) channels are activated normally in gpa1 guard cells. In addition, H(2)O(2) inhibition of stomatal opening and promotion of stomatal closure are not disrupted in gpa1 mutant guard cells. These data indicate that absence of GPA1 interrupts ABA signalling between ABA reception and ROS production, with a consequent impairment in Ca(2+)-channel activation.

Salmonella enterica subsp. enterica serovar Typhimurium was isolated from diarrheic piglets in 2 periods, 2000-2001 (n = 25) and 2005-2006 (n = 17). To compare the characteristics of the isolates collected during the 2 periods, all isolates were tested for antimicrobial resistance, the presence of virulence genes, and pulsed-field gel electrophoresis (PFGE) patterns. All 42 isolates were resistant to at least 1 of the 20 antimicrobials tested, and 39 (93%) were resistant to 2 or more antimicrobials. One isolate was resistant to 12 antimicrobials. Profiles of antimicrobial resistance revealed 20 resistance types. Several isolates were also resistant to quinolones and expanded-spectrum cephalosporins. Ten isolates (24%) were resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT); only one isolate had been isolated in 2000-2001, indicating that this type of resistance has rapidly disseminated. Polymerase chain reaction (PCR) assays revealed that all the isolates carried invA. Among the 25 strains isolated in 2000-2001, all carried the sipA, sopA, sopD, sopE2, and ssaR genes, and 96% carried sopB and sifA. Among the 17 strains isolated in 2005-2006, all carried sifA, and approximately 90% carried sipA, sopA, sopB, sopD, sopE2, and ssaR. However, only 6 (14%) of the 42 isolates carried spvC. By PFGE analysis, all 42 strains were classified into 4 major clusters, basically by collection period. The genetic similarity according to PFGE suggests that the strains isolated from diarrheic piglets of this region within the same period may be closely related.

Annexins are Ca2+--and phospholipid-binding proteins that form an evolutionarily conserved multigene family throughout the animal and plant kingdoms. Two annexins, AnnAt1 and AnnAt4, have been identified as components in osmotic stress and abscisic acid signaling in Arabidopsis. Here, we report that AnnAt1 and AnnAt4 regulate plant stress responses in a light-dependent manner. The single-mutant annAt1 and annAt4 plants showed tolerance to drought and salt stress, which was greatly enhanced in double-mutant annAt1annAt4 plants, but AnnAt4-overexpressing transgenic plants (35S:AnnAt4) were more sensitive to stress treatments under long day conditions. Furthermore, expression of stress-related genes was altered in these mutant and transgenic plants. Upon dehydration and salt treatment, AtNCED3, encoding 9-cis-epoxycarotenoid dioxygenase, and P5CS1, encoding Δ-1-pyrroline-5-carboxylate synthase, which are key enzymes in ABA and proline synthesis, respectively, were highly induced in annAt1annAt4 plants and to a lesser extent in annAt1 and annAt4 plants, but not in 35S:AnnAt4 plants. While annAt1 plants were more drought sensitive, annAt4 plants were more tolerant in short days than in long days. In vitro and in vivo binding assays revealed that AnnAt1 and AnnAt4 bind to each other in a Ca2+-dependent manner. Our results suggest that AnnAt1 and AnnAt4 function cooperatively in response to drought and salt stress and their functions are affected by photoperiod.

Phosphatidylinositol 3-kinases (PtdIns 3-kinases) that produce phosphatidylinositol (3,4,5) triphosphate (PtdIns(3,4,5)P(3)) are considered to be important regulators of actin dynamics in animal cells. In plants, neither PtdIns(3,4,5)P(3) nor the enzyme that produces this lipid has been reported. However, a PtdIns 3-kinase that produces phosphatidylinositol 3-phosphate (PtdIns3P) has been identified, suggesting that PtdIns3P, instead of PtdIns(3,4,5)P(3), regulates actin dynamics in plant cells. Phosphatidylinositol 4-kinase (PtdIns 4-kinase) is closely associated with the actin cytoskeleton in plant cells, suggesting a role for this lipid kinase and its product phosphatidylinositol 4-phosphate (PtdIns4P) in actin-related processes. Here, we investigated whether or not PtdIns3P or PtdIns4P plays a role in actin reorganization induced by a plant hormone abscisic acid (ABA) in guard cells of day flower (Commelina communis). ABA-induced changes in actin filaments were inhibited by LY294002 (LY) and wortmannin (WM), inhibitors of PtdIns3P and PtdIns4P synthesis. Expression of PtdIns3P- and PtdIns4P-binding domains also inhibited ABA-induced actin reorganization in a manner similar to LY and WM. These results suggest that PtdIns3P and PtdIns4P regulate actin dynamics in guard cells. Furthermore, we demonstrate that PtdIns3P exerts its effect on actin dynamics, at least in part, via generation of reactive oxygen species (ROS) in response to ABA.

ROP small G proteins function as molecular switches in diverse signaling processes. Here, we investigated signals that activate ROP2 in guard cells. In guard cells of Vicia faba expressing Arabidopsis thaliana constitutively active (CA) ROP2 fused to red fluorescent protein (RFP-CA-ROP2), fluorescence localized exclusively at the plasma membrane, whereas a dominant negative version of RFP-ROP2 (DN-ROP2) localized in the cytoplasm. In guard cells expressing green fluorescent protein-ROP2, the relative fluorescence intensity at the plasma membrane increased upon illumination, suggesting that light activates ROP2. Unlike previously reported light-activated factors, light-activated ROP2 inhibits rather than accelerates light-induced stomatal opening; stomata bordered by guard cells transformed with CA-rop2 opened less than controls upon light irradiation. When introduced into guard cells together with CA-ROP2, At RhoGDI1, which encodes a guanine nucleotide dissociation inhibitor, inhibited plasma membrane localization of CA-ROP2 and abolished the inhibitory effect of CA-ROP2 on light-induced stomatal opening, supporting the negative effect of active ROP2 on stomatal opening. Mutant rop2 Arabidopsis guard cells showed phenotypes similar to those of transformed V. faba guard cells; CA-rop2 stomata opened more slowly and to a lesser extent, and DN-rop2 stomata opened faster than wild-type stomata in response to light. Moreover, in rop2 knockout plants, stomata opened faster and to a greater extent than wild-type stomata in response to light. Thus, ROP2 is a light-activated negative factor that attenuates the extent of light-induced changes in stomatal aperture. The inhibition of light-induced stomatal opening by light-activated ROP2 suggests the existence of feedback regulatory mechanisms through which stomatal apertures may be finely controlled.

Previously, we demonstrated that a protein that binds phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] inhibits both light-induced stomatal opening and ABA-induced stomatal closing. The latter effect is due to a reduction in free PtdIns(4,5)P(2), decreasing production of inositol 1,4,5-trisphosphate and phosphatidic acid by phospholipases C and D. However, it is less clear how PtdIns(4,5)P(2) modulates stomatal opening. We found that in response to white light irradiation, the PtdIns(4,5)P(2)-binding domain GFP:PLCdelta1PH translocated from the cytosol into the plasma membrane. This suggests that the level of PtdIns(4,5)P(2) increases at the plasma membrane upon illumination. Exogenously administered PtdIns(4,5)P(2) substituted for light stimuli, inducing stomatal opening and swelling of guard cell protoplasts. To identify PtdIns(4,5)P(2) targets we performed patch-clamp experiments, and found that anion channel activity was inhibited by PtdIns(4,5)P(2). Genetic analyses using an Arabidopsis PIP5K4 mutant further supported the role of PtdIns(4,5)P(2) in stomatal opening. The reduced stomatal opening movements exhibited by a mutant of Arabidopsis PIP5K4 (At3g56960) was countered by exogenous application of PtdIns(4,5)P(2). The phenotype of reduced stomatal opening in the pip5k4 mutant was recovered in lines complemented with the full-length PIP5K4. Together, these data suggest that PIP5K4 produces PtdIns(4,5)P(2) in irradiated guard cells, inhibiting anion channels to allow full stomatal opening.

Enterohemorrhagic Escherichia coli (EHEC) is an important cause of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome worldwide. E. coli O26 and O111 are the serotypes most frequently isolated from human EHEC infections in Korea. Cattle are considered to be the major sources of E. coli O26 and O111. This study investigated the prevalence of E. coli O26 and O111 in fecal samples from cattle in Korea from April 2002 to March 2004. Out of 809 samples, 54 (6.67%), 37 (4.57%), and 16 (1.98%) tested positive for O26, O111, and both O26 and O111, respectively. Most of the E. coli O26 and O111 strains were isolated from May to October of each year. PCR analysis of the EHEC virulence markers revealed that most of the E. coli O26 and O111 isolates were positive for ehxA, eaeA and stx1 and/or stx2. These results suggest that the majority of Korean E. coli O26 and O111 isolates from cattle can cause serious diseases in humans.

Actinomycin D (act-D) anchors itself into DNA-base pairs by intercalation and thereby inhibits mRNA synthesis. It has been well established that act-D elicits apoptosis in various cell types involving endothelial cells. However, the regulatory mechanisms of actinomycin D-induced apoptotic cell death still remain unclear. Here, we investigated apoptotic cell death and its underlying regulatory mechanisms elicited by actinomycin D in ECV304. Act-D induced typical apoptotic features including chromatin condensation and translocation of phosphatidylserine. Since the phosphoinositide 3-OH kinase (PI3K)/protein kinase B (PKB) signaling pathway has been shown to prevent apoptosis in various cell types, it was of interest to determine if this pathway could protect against apoptosis induced by act-D. Inhibition of PI3K/PKB significantly increased act-D-induced apoptosis. Moreover, act-D-induced cell death was physiologically linked to PKB-mediated cell survival through caspase-8. These results suggest that cross-talk between the PKB and caspase-8 pathways may regulate the balance between cell survival and cell death in ECV304.

Apoptosis is a common mode of programmed cell death in multicellular organisms. However, the recent observation of yeast cell death displaying the morphology of apoptosis has suggested the presence of an ancestral cell death machinery. Here we examined apoptotic features induced by reactive oxygen species (ROS) in yeast. Saccharomyces cerevisiae show typical apoptotic features upon exposure to ROS: membrane staining with annexin V and DNA fragmentation by the TUNEL assay. The detection of apoptotic features in yeast strongly support the existence of molecular machinery performing the basic pathways of apoptosis. The phosphoinositide 3-OH kinase (PI3K)/protein kinase B (PKB) signaling pathway has been shown to prevent apoptosis in a variety of cells. It is therefore of interest to determine whether the PI3K/PKB signaling pathway is capable of protecting yeast from apoptosis induced by ROS. We determined that PI3K/PKB is capable of significantly inhibiting ROS-evoked apoptosis in yeast. These results suggest that yeast may provide a suitable model system in which to study the apoptotic signaling pathway elicited by a variety of stimuli.