AtNUDX6, an ADP-Ribose/NADH Pyrophosphohydrolase in Arabidopsis,Positively Regulates NPR1-Dependent Salicylic Acid Signaling

Here, we investigated the physiological role of Arabidopsis (Arabidopsis thaliana) AtNUDX6, the gene encoding ADP-ribose (Rib)/NADH pyrophosphohydrolase, using its overexpressor (Pro_35S:AtNUDX6) or disruptant (KO-nudx6). The level of NADH in Pro_35S:AtNUDX6 and KO-nudx6 plants was decreased and increased, respectively, compared with that of the control plants, while the level of ADP-Rib was not changed in either plant. The activity of pyrophosphohydrolase toward NADH was enhanced and reduced in the Pro_35S:AtNUDX6 and KO-nudx6 plants, respectively. The decrease in the activity of NADH pyrophosphohydrolase and the increase in the level of NADH were observed in the rosette and cauline leaves, but not in the roots, of the KO-nudx6 plants. Notably, the expression level of AtNUDX6 and the activity of NADH pyrophosphohydrolase in the control plants, but not in the KO-nudx6 plants, were increased by the treatment with salicylic acid (SA). The expression of SA-induced genes (PR1, WRKY70, NIMIN1, and NIMIN2) depending on NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1), a key component required for pathogen resistance, was significantly suppressed and enhanced in the KO-nudx6 and Pro_35S:AtNUDX6 plants, respectively, under the treatment with SA. Induction of thioredoxin h5 (TRX-h5) expression, which catalyzes a SA-induced NPR1 activation, was suppressed and accelerated in the KO-nudx6 and Pro_35S:AtNUDX6 plants, respectively. The expression of isochorismate synthase1, required for the regulation of SA synthesis through the NPR1-mediated feedback loop, was decreased and increased in the KO-nudx6 and Pro_35S:AtNUDX6 plants, respectively. Judging from seed germination rates, the KO-nudx6 plants had enhanced sensitivity to the toxicity of high-level SA. These results indicated that AtNUDX6 is a modulator of NADH rather than ADP-Rib metabolism and that, through induction of TRX-h5 expression, AtNUDX6 significantly impacts the plant immune response as a positive regulator of NPR1-dependent SA signaling pathways.Nudix (nucleoside diphosphates linked to some moiety X) hydrolases are a phylogenetically widespread enzyme family and are widely distributed among all classes of organisms, such as bacteria, yeast, algae, nematodes, vertebrates, and plants (Bessman et al., 1996; Xu et al., 2004; Kraszewska, 2008). The enzymes catalyze, with varying degrees of substrate specificity, the hydrolysis of a variety of nucleoside diphosphate derivatives: nucleoside diphosphates and triphosphates and their oxidized forms, dinucleoside polyphosphates, nucleotide sugars, NADH, CoA, and the mRNA caps (McLennan, 2006; Kraszewska, 2008; Gunawardana et al., 2009). Since these compounds are often toxic to cells, Nudix hydrolases seem to play protective, regulatory, and signaling roles in metabolism by hydrolytically removing such compounds (Bessman et al., 1996; Xu et al., 2004).We reported the molecular and enzymatic characteristics of Nudix hydrolases (AtNUDX1–AtNUDX27) in Arabidopsis (Arabidopsis thaliana) plants (Ogawa et al., 2005, 2008). Notably, among 27 types of AtNUDXs, cytosolic AtNUDX2, AtNUDX6, AtNUDX7, and AtNUDX10 had pyrophosphohydrolase activity toward both ADP-Rib and NADH in vitro. Recent studies have shown that the actions of NADH and/or ADP-Rib pyrophosphohydrolases are closely related to defense systems in response to biotic and abiotic stresses in higher plants.It has been reported that the expression of AtNUDX7 is induced by avirulent pathogenic attacks. Knockout AtNUDX7 mutants (KO-nudx7) showed enhanced resistance against both virulent and avirulent bacterial strains (Bartsch et al., 2006; Jambunathan and Mahalingam, 2006; Adams-Phillips et al., 2008). In addition, it was revealed that AtNUDX7 functions as a negative regulator on ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) signaling required for basal resistance to invasive pathogens (Bartsch et al., 2006); EDS1 regulates accumulation of the phenolic defense molecule, salicylic acid (SA), and other as yet unidentified signal intermediates and controls the defense activation and programmed cell death by collaborating with its interaction partner PHYTOALEXIN-DEFICIENT4 in cells surrounding pathogen infection foci. Furthermore, Ge et al. (2007) reported that AtNUDX7 functions to prevent excessive stimulation of the defense response, which is dependent on and independent of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1), a master regulator of SA-induced defense genes (SAIGs), and SA accumulation.On the other hand, we recently demonstrated the roles of Arabidopsis NADH/ADP-Rib pyrophosphohydrolases (AtNUDX2 and AtNUDX7) in tolerance to oxidative stress using the respective overexpressors (Pro_35S:AtNUDX2 and Pro_35S:AtNUDX7) or disruptants (KO-nudx7; Ishikawa et al., 2009; Ogawa et al., 2009). Interestingly, overexpression of AtNUDX2 and AtNUDX7 in Arabidopsis plants was responsible for an enhanced tolerance to oxidative stress derived from the treatment with paraquat (an agent producing O₂⁻) and salinity. Taken together, these results revealed that both AtNUDX2 and AtNUDX7 function in accelerating nucleotide recycling from ADP-Rib produced by poly(ADP-Rib) metabolism, leading to suppression of the overconsumption of NAD⁺ and ATP in Arabidopsis cells under stressful conditions. In addition, AtNUDX7 served to balance between NADH and NAD⁺ by NADH turnover and to regulate the defense mechanisms against DNA damage by modulation of the poly(ADP-ribosyl)ation (PAR) reaction through NADH metabolism in response to oxidative stress (Ishikawa et al., 2009; Ogawa et al., 2009). These findings clearly indicated that the regulation of NADH and/or ADP-Rib metabolism via Nudix hydrolases is involved in the responses to both biotic and abiotic stresses in higher plants.The question that we must consider next is whether the other AtNUDXs (AtNUDX6 and AtNUDX10) with pyrophosphohydrolase activities toward ADP-Rib and NADH are involved in the defense systems against oxidative stress and pathogen attack. The expression of AtNUDX6 has been reported to be induced by pathogenic attacks and treatment with the SA analogs 2,6-dichloroisonicotinic acid and acibenzolar-S-methyl benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH; Bartsch et al., 2006; Qiu et al., 2008; Knoth et al., 2009). Furthermore, the expression of AtNUDX6 was strongly dependent on EDS1 (Bartsch et al., 2006). However, the functional significance of AtNUDX6 is still unclear, since a loss-of-function mutant of AtNUDX6 has not yet been found.In this paper, to assess the physiological function of AtNUDX6, we identified an Arabidopsis mutant in which T-DNA is inserted into AtNUDX6 and subsequently studied the levels of ADP-Rib and NAD(H), PAR activity, expression of genes related to SA signaling, and SA tolerance in the AtNUDX6 overexpressors and disruptants in comparison with the AtNUDX7 disruptants. The results obtained here indicated that AtNUDX6 positively regulates NPR1-dependent SA signaling via modulation of NADH metabolism in the plant immune response.     

Modulation of the Poly(ADP-ribosyl)ation Reaction via the Arabidopsis ADP-Ribose/NADH Pyrophosphohydrolase,AtNUDX7, Is Involved in the Response to Oxidative Stress

Here, we assessed modulation of the poly(ADP-ribosyl)ation (PAR) reaction by an Arabidopsis (Arabidopsis thaliana) ADP-ribose (Rib)/NADH pyrophosphohydrolase, AtNUDX7 (for Arabidopsis Nudix hydrolase 7), in AtNUDX7-overexpressed (Pro_35S:AtNUDX7) or AtNUDX7-disrupted (KO-nudx7) plants under normal conditions and oxidative stress caused by paraquat treatment. Levels of NADH and ADP-Rib were decreased in the Pro_35S:AtNUDX7 plants but increased in the KO-nudx7 plants under normal conditions and oxidative stress compared with the control plants, indicating that AtNUDX7 hydrolyzes both ADP-Rib and NADH as physiological substrates. The Pro_35S:AtNUDX7 and KO-nudx7 plants showed increased and decreased tolerance, respectively, to oxidative stress compared with the control plants. Levels of poly(ADP-Rib) in the Pro_35S:AtNUDX7 and KO-nudx7 plants were markedly higher and lower, respectively, than those in the control plants. Depletion of NAD⁺ and ATP resulting from the activation of the PAR reaction under oxidative stress was completely suppressed in the Pro_35S:AtNUDX7 plants. Accumulation of NAD⁺ and ATP was observed in the KO-nudx7- and 3-aminobenzamide-treated plants, in which the PAR reaction was suppressed. The expression levels of DNA repair factors, AtXRCC1 and AtXRCC2 (for x-ray repair cross-complementing factors 1 and 2), paralleled that of AtNUDX7 under both normal conditions and oxidative stress, although an inverse correlation was observed between the levels of AtXRCC3, AtRAD51 (for Escherichia coli RecA homolog), AtDMC1 (for disrupted meiotic cDNA), and AtMND1 (for meiotic nuclear divisions) and AtNUDX7. These findings suggest that AtNUDX7 controls the balance between NADH and NAD⁺ by NADH turnover under normal conditions. Under oxidative stress, AtNUDX7 serves to maintain NAD⁺ levels by supplying ATP via nucleotide recycling from free ADP-Rib molecules and thus regulates the defense mechanisms against oxidative DNA damage via modulation of the PAR reaction.Reactive oxygen species (ROS) are by-products of normal metabolic processes, including chloroplastic, mitochondrial, and plasma membrane-linked electron transport systems, in all aerobic organisms (Gutteridge and Halliwell, 1989). Although the production and destruction of ROS are in balance, the imposition of biotic and abiotic stressful conditions can give rise to excess concentrations of ROS, leading to an imbalance of production and scavenging mechanisms (Mittler, 2002; Mullineaux and Karpinski, 2002; Kroj et al., 2003; Mahalingam et al., 2003). Excess ROS, leading to oxidative stress, can damage organelles, oxidize proteins, nick DNA (single-base DNA damage), deplete antioxidant levels, and ultimately trigger cell death (Gutteridge and Halliwell, 1989). Recently, ROS have been recognized as important signaling molecules that control diverse signaling pathways involved in a variety of cellular responses such as programmed cell death, pathogen defense, and hormone signaling (Foyer and Noctor, 2005; Kwak et al., 2006; Torres et al., 2006). In addition, oxidative stress causes dramatic inhibition of the tricarboxylic acid cycle and large sectors of amino acid metabolism followed by backing up of glycolysis and diversion of carbon into the oxidative pentose phosphate pathway (Baxter et al., 2007). Therefore, organisms have developed efficient systems to keep ROS levels in check and repair damage from attack by ROS.Among various defense systems against attack by ROS, the poly(ADP-ribosyl)ation (PAR) of proteins by poly(ADP-Rib)polymerase (PARP), by which branched polymers of ADP-Rib are attached using β-NAD⁺ to a specific amino acid residue of an acceptor protein, is a posttranslational modification for responding early to DNA damage, such as single-strand DNA break and resealing, caused by oxidative stress and, thus, is crucial for genomic integrity and cell survival (Qin et al., 2008). PARP detects DNA strand breaks and converts the damage into intracellular signals that can activate DNA repair programs or cell death, according to the severity of the injury, via the PAR reaction of nuclear proteins involved in chromatin architecture and DNA metabolism and interacts with the x-ray repair cross complementing factor 1 (XRCC1), an adaptor protein that also has two interfaces with two important single-strand DNA break (SSB) repair (SSBR)/base excision repair (BER) enzymes: DNA ligase and DNA polymerase β (Caldecott et al., 1995, 1996; Kubota et al., 1996; Masson et al., 1998). DNA polymerase β fills the single nucleotide gap, preparing the strand for ligation by a complex of DNA ligase III and XRCC1 (Winters et al., 1999; Thompson and West, 2000). Thereby, the fast recruitment of SSBR/BER factors is archived in the site of the lesion. Modifications of proteins with poly(ADP-Rib) are reversed by poly(ADP-Rib) glycohydrolase (PARG), by which ADP-Rib polymers are hydrolyzed to free ADP-Rib, since incorrect signal transduction is caused by excessive accumulation of poly(ADP-Rib) modification (Davidovic et al., 2001). However, it has been reported that a massive PAR reaction results in the overconsumption of NAD⁺ and ATP and, ultimately, in energy depletion causing necrotic cell death (Ha and Snyder, 1999; Virág and Szabó, 2002; De Block et al., 2005).Nudix (for nucleoside diphosphates linked to some moiety X) hydrolases catalyze the hydrolysis of intact and oxidatively damaged nucleoside diphosphates and triphosphates, nucleotide sugars, coenzymes, dinucleoside polyphosphates, and RNA caps in various organisms such as bacteria, yeast, algae, nematodes, vertebrates, and plants (Bessman et al., 1996; Xu et al., 2004; Kraszewska, 2008). We have previously reported the characteristics of cytosolic Nudix hydrolases (AtNUDX1–AtNUDX11) in Arabidopsis (Arabidopsis thaliana; Ogawa et al., 2005). Among them, the recombinant AtNUDX7 showed high affinity for ADP-Rib and NADH as substrates in vitro, converting NADH to a reduced form of nicotinamide mononucleotide (NMNH) plus AMP and ADP-Rib to AMP plus Rib 5-P (Ogawa et al., 2005). AtNUDX7 was expressed more strongly in leaf than in stem and root. Therefore, the enzyme might be involved in nucleotide recycling relating to the metabolism of NADH and/or poly(ADP-Rib).Recent studies revealed that the actions of AtNUDX7 (At4g12720) are closely related to immune responses to pathogens. Knockout of AtNUDX7 (KO-nudx7) in Arabidopsis plants led to deleterious inference for cells, such as microscopic cell death, constitutive expression of pathogenesis-related genes, resistance to bacterial pathogens, and accumulation of NADH (Jambunathan and Mahalingam, 2006). Furthermore, AtNUDX7 exerted a negative regulatory effect on EDS1 signaling, which controls the activation of defenses and programmed cell death conditioned by intracellular Toll-related immune receptors that recognized specific pathogen effectors (Bartsch et al., 2006). More recently, Ge et al. (2007) reported that KO-nudx7 plants show heightened defense responses, which are both dependent on and independent of the accumulation of NPR1 and salicylic acid, to pathogenic attack. On the other hand, Adams-Phillips et al. (2008) reported that KO-nudx7 plants exhibit a reduced hypersensitive-response phenotype, although the growth of both virulent and avirulent pathogens is suppressed in the plants. These findings support the hypothesis that regulation of the metabolism of NADH and/or ADP-Rib by Nudix hydrolases is important for stress-related defense systems in higher plants. However, the direct actions of the enzymes on stress responses are not established yet.In this study, to assess the functions of Arabidopsis Nudix hydrolases having ADP-Rib and NADH pyrophosphohydrolase activities under normal conditions and oxidative stress, we analyzed the effect of the overexpression or disruption of AtNUDX7 on levels of ADP-Rib, NAD(H), and ATP as well as PAR activity and oxidative stress tolerance in Arabidopsis. The evidence presented here suggests that AtNUDX7 serves to balance between NADH and NAD⁺ by NADH turnover under normal conditions. In addition, AtNUDX7 functions in the maintenance of NAD⁺ levels by supplying ATP via nucleotide recycling from free ADP-Rib molecules and the modulation of the PAR reaction, thereby regulating the DNA repair pathways, in response to oxidative stress.     

Plant defensins: Defense,development and application

Plant defensins are small, highly stable, cysteine-rich peptides that constitute a part of the innate immune system primarily directed against fungal pathogens. Biological activities reported for plant defensins include antifungal activity, antibacterial activity, proteinase inhibitory activity and insect amylase inhibitory activity. Plant defensins have been shown to inhibit infectious diseases of humans and to induce apoptosis in a human pathogen. Transgenic plants overexpressing defensins are strongly resistant to fungal pathogens. Based on recent studies, some plant defensins are not merely toxic to microbes but also have roles in regulating plant growth and development.Key words: defensin, antifungal, antimicrobial peptide, development, innate immunityDefensins are diverse members of a large family of cationic host defence peptides (HDP), widely distributed throughout the plant and animal kingdoms.^1–3 Defensins and defensin-like peptides are functionally diverse, disrupting microbial membranes and acting as ligands for cellular recognition and signaling.⁴ In the early 1990s, the first members of the family of plant defensins were isolated from wheat and barley grains.^5,6 Those proteins were originally called γ-thionins because their size (∼5 kDa, 45 to 54 amino acids) and cysteine content (typically 4, 6 or 8 cysteine residues) were found to be similar to the thionins.⁷ Subsequent “γ-thionins” homologous proteins were indentified and cDNAs were cloned from various monocot or dicot seeds.⁸ Terras and his colleagues⁹ isolated two antifungal peptides, Rs-AFP1 and Rs-AFP2, noticed that the plant peptides'' structural and functional properties resemble those of insect and mammalian defensins, and therefore termed the family of peptides “plant defensins” in 1995. Sequences of more than 80 different plant defensin genes from different plant species were analyzed.¹⁰ A query of the UniProt database (www.uniprot.org/) currently reveals publications of 371 plant defensins available for review. The Arabidopsis genome alone contains more than 300 defensin-like (DEFL) peptides, 78% of which have a cysteine-stabilized α-helix β-sheet (CSαβ) motif common to plant and invertebrate defensins.¹¹ In addition, over 1,000 DEFL genes have been identified from plant EST projects.¹²Unlike the insect and mammalian defensins, which are mainly active against bacteria,^2,3,10,13 plant defensins, with a few exceptions, do not have antibacterial activity.¹⁴ Most plant defensins are involved in defense against a broad range of fungi.^2,3,10,15 They are not only active against phytopathogenic fungi (such as Fusarium culmorum and Botrytis cinerea), but also against baker''s yeast and human pathogenic fungi (such as Candida albicans).² Plant defensins have also been shown to inhibit the growth of roots and root hairs in Arabidopsis thaliana¹⁶ and alter growth of various tomato organs which can assume multiple functions related to defense and development.⁴     

Hypoxia: A novel function for VIN3

VERNALIZATION INSENSITIVE 3 (VIN3) encodes a PHD domain chromatin remodelling protein that is induced in response to cold and is required for the establishment of the vernalization response in Arabidopsis thaliana.¹ Vernalization is the acquisition of the competence to flower after exposure to prolonged low temperatures, which in Arabidopsis is associated with the epigenetic repression of the floral repressor FLOWERING LOCUS C (FLC).^2,3 During vernalization VIN3 binds to the chromatin of the FLC locus,¹ and interacts with conserved components of Polycomb-group Repressive Complex 2 (PRC2).^4,5 This complex catalyses the tri-methylation of histone H3 lysine 27 (H3K27me3),^4,6,7 a repressive chromatin mark that increases at the FLC locus as a result of vernalization.^4,7–10 In our recent paper¹¹ we found that VIN3 is also induced by hypoxic conditions, and as is the case with low temperatures, induction occurs in a quantitative manner. Our experiments indicated that VIN3 is required for the survival of Arabidopsis seedlings exposed to low oxygen conditions. We suggested that the function of VIN3 during low oxygen conditions is likely to involve the mediation of chromatin modifications at certain loci that help the survival of Arabidopsis in response to prolonged hypoxia. Here we discuss the implications of our observations and hypotheses in terms of epigenetic mechanisms controlling gene regulation in response to hypoxia.Key words: arabidopsis, VIN3, FLC, hypoxia, vernalization, chromatin remodelling, survival     

Cell Migration from Baby to Mother

Fetal cells migrate into the mother during pregnancy. Fetomaternal transfer probably occurs in all pregnancies and in humans the fetal cells can persist for decades. Microchimeric fetal cells are found in various maternal tissues and organs including blood, bone marrow, skin and liver. In mice, fetal cells have also been found in the brain. The fetal cells also appear to target sites of injury. Fetomaternal microchimerism may have important implications for the immune status of women, influencing autoimmunity and tolerance to transplants. Further understanding of the ability of fetal cells to cross both the placental and blood-brain barriers, to migrate into diverse tissues, and to differentiate into multiple cell types may also advance strategies for intravenous transplantation of stem cells for cytotherapeutic repair. Here we discuss hypotheses for how fetal cells cross the placental and blood-brain barriers and the persistence and distribution of fetal cells in the mother.Key Words: fetomaternal microchimerism, stem cells, progenitor cells, placental barrier, blood-brain barrier, adhesion, migrationMicrochimerism is the presence of a small population of genetically distinct and separately derived cells within an individual. This commonly occurs following transfusion or transplantation.^1–3 Microchimerism can also occur between mother and fetus. Small numbers of cells traffic across the placenta during pregnancy. This exchange occurs both from the fetus to the mother (fetomaternal)^4–7 and from the mother to the fetus.^8–10 Similar exchange may also occur between monochorionic twins in utero.^11–13 There is increasing evidence that fetomaternal microchimerism persists lifelong in many child-bearing women.^7,14 The significance of fetomaternal microchimerism remains unclear. It could be that fetomaternal microchimerism is an epiphenomenon of pregnancy. Alternatively, it could be a mechanism by which the fetus ensures maternal fitness in order to enhance its own chances of survival. In either case, the occurrence of pregnancy-acquired microchimerism in women may have implications for graft survival and autoimmunity. More detailed understanding of the biology of microchimeric fetal cells may also advance progress towards cytotherapeutic repair via intravenous transplantation of stem or progenitor cells.Trophoblasts were the first zygote-derived cell type found to cross into the mother. In 1893, Schmorl reported the appearance of trophoblasts in the maternal pulmonary vasculature.¹⁵ Later, trophoblasts were also observed in the maternal circulation.^16–20 Subsequently various other fetal cell types derived from fetal blood were also found in the maternal circulation.^21,22 These fetal cell types included lymphocytes,²³ erythroblasts or nucleated red blood cells,^24,25 haematopoietic progenitors^7,26,27 and putative mesenchymal progenitors.^14,28 While it has been suggested that small numbers of fetal cells traffic across the placenta in every human pregnancy,^29–31 trophoblast release does not appear to occur in all pregnancies.³² Likewise, in mice, fetal cells have also been reported in maternal blood.^33,34 In the mouse, fetomaternal transfer also appears to occur during all pregnancies.³⁵     

RALFs: Peptide regulators of plant growth

Peptide signaling regulates a variety of developmental processes and environmental responses in plants.^1–6 For example, the peptide systemin induces the systemic defense response in tomato⁷ and defensins are small cysteine-rich proteins that are involved in the innate immune system of plants.^8,9 The CLAVATA3 peptide regulates meristem size¹⁰ and the SCR peptide is the pollen self-incompatibility recognition factor in the Brassicaceae.^11,12 LURE peptides produced by synergid cells attract pollen tubes to the embryo sac.⁹ RALFs are a recently discovered family of plant peptides that play a role in plant cell growth.Key words: peptide, growth factor, alkalinization     

Complete Genome Sequence of Croceibacter atlanticus HTCC2559T

Here we announce the complete genome sequence of Croceibacter atlanticus HTCC2559^T, which was isolated by high-throughput dilution-to-extinction culturing from the Bermuda Atlantic Time Series station in the Western Sargasso Sea. Strain HTCC2559^T contained genes for carotenoid biosynthesis, flavonoid biosynthesis, and several macromolecule-degrading enzymes. The genome confirmed physiological observations of cultivated Croceibacter atlanticus strain HTCC2559^T, which identified it as an obligate chemoheterotroph.The phylum Bacteroidetes comprises 6 to ∼30% of total bacterial communities in the ocean by fluorescence in situ hybridization (8-10). Most marine Bacteroidetes are in the family Flavobacteriaceae, most of which are aerobic respiratory heterotrophs that form a well-defined clade by 16S rRNA phylogenetic analyses (4). The members of this family are well known for degrading macromolecules, including chitin, DNA, cellulose, starch, and pectin (17), suggesting their environmental roles as detritus decomposers in the ocean (6). Marine Polaribacter and Dokdonia species in the Flavobacteriaceae have also shown to have photoheterotrophic metabolism mediated by proteorhodopsins (11, 12).Several strains of the family Flavobacteriaceae were isolated from the Sargasso Sea and Oregon coast, using high-throughput culturing approaches (7). Croceibacter atlanticus HTCC2559^T was cultivated from seawater collected at a depth of 250 m from the Sargasso Sea and was identified as a new genus in the family Flavobacteriaceae based on its 16S rRNA gene sequence similarities (6). Strain HTCC2559^T met the minimal standards for genera of the family Flavobacteriaceae (3) on the basis of phenotypic characteristics (6).Here we report the complete genome sequence of Croceibacter atlanticus HTCC2559^T. The genome sequencing was initiated by the J. Craig Venter Institute as a part of the Moore Foundation Microbial Genome Sequencing Project and completed in the current announcement. Gaps among contigs were closed by Genotech Co., Ltd. (Daejeon, Korea), using direct sequencing of combinatorial PCR products (16). The HTCC2559^T genome was analyzed with a genome annotation system based on GenDB (14) at Oregon State University and with the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (15, 16).The HTCC2559^T genome is 2,952,962 bp long, with 33.9 mol% G+C content, and there was no evidence of plasmids. The number of protein-coding genes was 2,715; there were two copies of the 16S-23S-5S rRNA operon and 36 tRNA genes. The HTCC2559^T genome contained genes for a complete tricarboxylic acid cycle, glycolysis, and a pentose phosphate pathway. The genome also contained sets of genes for metabolic enzymes involved in carotenoid biosynthesis and also a serine/glycine hydroxymethyltransferase, which is often associated with the assimilatory serine cycle (13). The potential for HTCC2559^T to use bacterial type III polyketide synthase (PKS) needs to be confirmed because this organism had a naringenin-chalcone synthase (CHS) or chalcone synthase (EC 2.3.1.74), a key enzyme in flavonoid biosynthesis. CHS initiates the addition of three molecules of malonyl coenzyme A (malonyl-CoA) to a starter CoA ester (e.g., 4-coumaroyl-CoA) (1) and takes part in a few bacterial type III polyketide synthase systems (1, 2, 5, 18).The complete genome sequence confirmed that strain HTCC2559^T is an obligate chemoheterotroph because no genes for phototrophy were found. As expected from physiological characteristics (6), the HTCC2559^T genome contained a set of genes coding for enzymes required to degrade high-molecular-weight compounds, including peptidases, metallo-/serine proteases, pectinase, alginate lyases, and α-amylase.     

Neutralization of Clostridium difficile toxin A using antibody combinations

The pathogenicity of Clostridium difficile (C. difficile) is mediated by the release of two toxins, A and B. Both toxins contain large clusters of repeats known as cell wall binding (CWB) domains responsible for binding epithelial cell surfaces. Several murine monoclonal antibodies were generated against the CWB domain of toxin A and screened for their ability to neutralize the toxin individually and in combination. Three antibodies capable of neutralizing toxin A all recognized multiple sites on toxin A, suggesting that the extent of surface coverage may contribute to neutralization. Combination of two noncompeting antibodies, denoted 3358 and 3359, enhanced toxin A neutralization over saturating levels of single antibodies. Antibody 3358 increased the level of detectable CWB domain on the surface of cells, while 3359 inhibited CWB domain cell surface association. These results suggest that antibody combinations that cover a broader epitope space on the CWB repeat domains of toxin A (and potentially toxin B) and utilize multiple mechanisms to reduce toxin internalization may provide enhanced protection against C. difficile-associated diarrhea.Key words: Clostridium difficile, toxin neutralization, therapeutic antibody, cell wall binding domains, repeat proteins, CROPs, mAb combinationThe most common cause of nosocomial antibiotic-associated diarrhea is the gram-positive, spore-forming anaerobic bacillus Clostridium difficile (C. difficile). Infection can be asymptomatic or lead to acute diarrhea, colitis, and in severe instances, pseudomembranous colitis and toxic megacolon.^1,2The pathological effects of C. difficile have long been linked to two secreted toxins, A and B.^3,4 Some strains, particularly the virulent and antibiotic-resistant strain 027 with toxinotype III, also produce a binary toxin whose significance in the pathogenicity and severity of disease is still unclear.⁵ Early studies including in vitro cell-killing assays and ex vivo models indicated that toxin A is more toxigenic than toxin B; however, recent gene manipulation studies and the emergence of virulent C. difficile strains that do not express significant levels of toxin A (termed “A⁻ B⁺”) suggest a critical role for toxin B in pathogenicity.^6,7Toxins A and B are large multidomain proteins with high homology to one another. The N-terminal region of both toxins enzymatically glucosylates small GTP binding proteins including Rho, Rac and CDC42,^8,9 leading to altered actin expression and the disruption of cytoskeletal integrity.^9,10 The C-terminal region of both toxins is composed of 20 to 30 residue repeats known as the clostridial repetitive oligopeptides (CROPs) or cell wall binding (CWB) domains due to their homology to the repeats of Streptococcus pneumoniae LytA,^11–14 and is responsible for cell surface recognition and endocytosis.^12,15–17C. difficile-associated diarrhea is often, but not always, induced by antibiotic clearance of the normal intestinal flora followed by mucosal C. difficile colonization resulting from preexisting antibiotic resistant C. difficile or concomitant exposure to C. difficile spores, particularly in hospitals. Treatments for C. difficile include administration of metronidazole or vancomycin.^2,18 These agents are effective; however, approximately 20% of patients relapse. Resistance of C. difficile to these antibiotics is also an emerging issue^19,20 and various non-antibiotic treatments are under investigation.^20–25Because hospital patients who contract C. difficile and remain asymptomatic have generally mounted strong antibody responses to the toxins,^26,27 active or passive immunization approaches are considered hopeful avenues of treatment for the disease. Toxins A and B have been the primary targets for immunization approaches.^20,28–33 Polyclonal antibodies against toxins A and B, particularly those that recognize the CWB domains, have been shown to effectively neutralize the toxins and inhibit morbidity in rodent infection models.³¹ Monoclonal antibodies (mAbs) against the CWB domains of the toxins have also demonstrated neutralizing capabilities; however, their activity in cell-based assays is significantly weaker than that observed for polyclonal antibody mixtures.^33–36We investigated the possibility of creating a cocktail of two or more neutralizing mAbs that target the CWB domain of toxin A with the goal of synthetically re-creating the superior neutralization properties of polyclonal antibody mixtures. Using the entire CWB domain of toxin A, antibodies were raised in rodents and screened for their ability to neutralize toxin A in a cell-based assay. Two mAbs, 3358 and 3359, that (1) both independently demonstrated marginal neutralization behavior and (2) did not cross-block one another from binding toxin A were identified. We report here that 3358 and 3359 use differing mechanisms to modify CWB-domain association with CHO cell surfaces and combine favorably to reduce toxin A-mediated cell lysis.     

Why have chloroplasts developed a unique motility system?

Organelle movement in plants is dependent on actin filaments with most of the organelles being transported along the actin cables by class XI myosins. Although chloroplast movement is also actin filament-dependent, a potential role of myosin motors in this process is poorly understood. Interestingly, chloroplasts can move in any direction and change the direction within short time periods, suggesting that chloroplasts use the newly formed actin filaments rather than preexisting actin cables. Furthermore, the data on myosin gene knockouts and knockdowns in Arabidopsis and tobacco do not support myosins'' XI role in chloroplast movement. Our recent studies revealed that chloroplast movement and positioning are mediated by the short actin filaments localized at chloroplast periphery (cp-actin filaments) rather than cytoplasmic actin cables. The accumulation of cp-actin filaments depends on kinesin-like proteins, KAC1 and KAC2, as well as on a chloroplast outer membrane protein CHUP1. We propose that plants evolved a myosin XI-independent mechanism of the actin-based chloroplast movement that is distinct from the mechanism used by other organelles.Key words: actin, Arabidopsis, blue light, kinesin, myosin, organelle movement, phototropinOrganelle movement and positioning are pivotal aspects of the intracellular dynamics in most eukaryotes. Although plants are sessile organisms, their organelles are quickly repositioned in response to fluctuating environmental conditions and certain endogenous signals. By and large, plant organelle movements and positioning are dependent on actin filaments, although microtubules play certain accessory roles in organelle dynamics.^1,2 Actin inhibitors effectively retard the movements of mitochondria,^3–6 peroxisomes,^5,7–11 Golgi stacks,^12,13 endoplasmic reticulum (ER),^14,15 and nuclei.^16–18 These organelles are co-aligned and associated with actin filaments.^{5,7,8,10–12,15,18} Recent progress in this field started to reveal the molecular motility system responsible for the organelle transport in plants.¹⁹Chloroplast movement is among the most fascinating models of organelle movement in plants because it is precisely controlled by ambient light conditions.^20,21 Weak light induces chloroplast accumulation response so that chloroplasts can capture photosynthetic light efficiently (Fig. 1A). Strong light induces chloroplast avoidance response to escape from photodamage (Fig. 1B).²² The blue light-induced chloroplast movement is mediated by the blue light receptor phototropin (phot). In some cryptogam plants, the red light-induced chloroplast movement is regulated by a chimeric phytochrome/phototropin photoreceptor neochrome.^23–25 In a model plant Arabidopsis, phot1 and phot2 function redundantly to regulate the accumulation response,²⁶ whereas phot2 alone is essential for the avoidance response.^27,28 Several additional factors regulating chloroplast movement were identified by analyses of Arabidopsis mutants deficient in chloroplast photorelocation.^29–32 In particular, identification of CHUP1 (chloroplast unusual positioning 1) revealed the connection between chloroplasts and actin filaments at the molecular level.²⁹ CHUP1 is a chloroplast outer membrane protein capable of interacting with F-actin, G-actin and profilin in vitro.^29,33,34 The chup1 mutant plants are defective in both the chloroplast movement and chloroplast anchorage to the plasma membrane,^22,29,33 suggesting that CHUP1 plays an important role in linking chloroplasts to the plasma membrane through the actin filaments. However, how chloroplasts move using the actin filaments and whether chloroplast movement utilizes the actin-based motility system similar to other organelle movements remained to be determined.Open in a separate windowFigure 1Schematic distribution patterns of chloroplasts in a palisade cell under different light conditions, weak (A) and strong (B) lights. Shown as a side view of mid-part of the cell and a top view with three different levels (i.e., top, middle and bottom of the cell). The cell was irradiated from the leaf surface shown as arrows. Weak light induces chloroplast accumulation response (A) and strong light induces the avoidance response (B).Here, we review the recent findings pointing to existence of a novel actin-based mechanisms for chloroplast movement and discuss the differences between the mechanism responsible for movement of chloroplasts and other organelles.     

Timing the switch to phototrophic growth: A possible role of GUN1

In young Arabidopsis seedlings, retrograde signaling from plastids regulates the expression of photosynthesis-associated nuclear genes in response to the developmental and functional state of the chloroplasts. The chloroplast-located PPR protein GUN1 is required for signalling following disruption of plastid protein synthesis early in seedling development before full photosynthetic competence has been achieved. Recently we showed that sucrose repression and the correct temporal expression of LHCB1, encoding a light-harvesting chlorophyll protein associated with photosystem II, are perturbed in gun1 mutant seedlings.¹ Additionally, we demonstrated that in gun1 seedlings anthocyanin accumulation and the expression of the “early” anthocyanin-biosynthesis genes is perturbed. Early seedling development, predominantly at the stage of hypocotyl elongation and cotyledon expansion, is also affected in gun1 seedlings in response to sucrose, ABA and disruption of plastid protein synthesis by lincomycin. These findings indicate a central role for GUN1 in plastid, sucrose and ABA signalling in early seedling development.Key words: ABA, ABI4, anthocyanin, chloroplast, GUN1, retrograde signalling, sucroseArabidopsis seedlings develop in response to light and other environmental cues. In young seedlings, development is fuelled by mobilization of lipid reserves until chloroplast biogenesis is complete and the seedlings can make the transition to phototrophic growth. The majority of proteins with functions related to photosynthesis are encoded by the nuclear genome, and their expression is coordinated with the expression of genes in the chloroplast genome. In developing seedlings, retrograde signaling from chloroplasts to the nucleus regulates the expression of these nuclear genes and is dependent on the developmental and functional status of the chloroplast. Two classes of gun (genomes uncoupled) mutants defective in retrograde signalling have been identified in Arabidopsis: the first, which comprises gun2–gun5, involves mutations in genes encoding components of tetrapyrrole biosynthesis.^2,3 The other comprises gun1, which has mutations in a nuclear gene encoding a plastid-located pentatricopeptide repeat (PPR) protein with an SMR (small MutS-related) domain near the C-terminus.^4,5 PPR proteins are known to have roles in RNA processing⁶ and the SMR domain of GUN1 has been shown to bind DNA,⁴ but the specific functions of these domains in GUN1 are not yet established. However, GUN1 has been shown to be involved in plastid gene expression-dependent,⁷ redox,⁴ ABA1,⁴ and sucrose signaling,^1,4,8 as well as light quality and intensity sensing pathways.^9–11 In addition, GUN1 has been shown to influence anthocyanin biosynthesis, hypocotyl extension and cotyledon expansion.^1,11     

Protein interactors of acyl-CoA-binding protein ACBP2 mediate cadmium tolerance in Arabidopsis

In our recent paper in the Plant Journal, we reported that Arabidopsis thaliana lysophospholipase 2 (lysoPL2) binds acyl-CoA-binding protein 2 (ACBP2) to mediate cadmium [Cd(II)] tolerance in transgenic Arabidopsis. ACBP2 contains ankyrin repeats that have been previously shown to mediate protein-protein interactions with an ethylene-responsive element binding protein (AtEBP) and a farnesylated protein 6 (AtFP6). Transgenic Arabidopsis ACBP2-overexpressors, lysoPL2-overexpressors and AtFP6-overexpressors all display enhanced Cd(II) tolerance, in comparison to wild type, suggesting that ACBP2 and its protein partners work together to mediate Cd(II) tolerance. Given that recombinant ACBP2 and AtFP6 can independently bind Cd(II) in vitro, they may be able to participate in Cd(II) translocation. The binding of recombinant ACBP2 to [¹⁴C]linoleoyl-CoA and [¹⁴C]linolenoyl-CoA implies its role in phospholipid repair. In conclusion, ACBP2 can mediate tolerance to Cd(II)-induced oxidative stress by interacting with two protein partners, AtFP6 and lysoPL2. Observations that ACBP2 also binds lysophosphatidylcholine (lysoPC) in vitro and that recombinant lysoPL2 degrades lysoPC, further confirm an interactive role for ACBP2 and lysoPL2 in overcoming Cd(II)-induced stress.Key words: acyl-CoA-binding protein, cadmium, hydrogen peroxide, lysophospholipase, oxidative stressAcyl-CoA-binding proteins (ACBP1 to ACBP6) are encoded by a multigene family in Arabidopsis thaliana.¹ These ACBP proteins are well studied in Arabidopsis in comparison to other organisms,^1–4 and are located in various subcellular compartments.¹ Plasma membranelocalized ACBP1 and ACBP2 contain ankyrin repeats that have been shown to function in protein-protein interactions.^5,6 ACBP1 and ACBP2 which share 76.9% amino acid identity also confer tolerance in transgenic Arabidopsis to lead [Pb(II)] and Cd(II), respectively.^1,5,7 Since recombinant ACBP1 and ACBP2 bind linolenoyl-CoA and linoleoyl-CoA in vitro, they may possibly be involved in phospholipid repair in response to heavy metal stress at the plasma membrane.^5,7 In contrast, ACBP3 is an extracellularly-localized protein⁸ while ACBP4, ACBP5 and ACBP6 are localized to cytosol.^9,10 ACBP1 and ACBP6 have recently been shown to be involved in freezing stress.^9,11 ACBP4 and ACBP5 bind oleoyl-CoA ester and their mRNA expressions are lightregulated.^12,13 Besides acyl-CoA esters, some ACBPs also bind phospholipids.^9,11,13 To investigate the biological function of ACBP2, we have proceeded to establish its interactors at the ankyrin repeats, including AtFP6,⁵ AtEBP⁶ and now lysoPL2 in the Plant Journal paper. While the significance in the interaction of ACBP2 with AtEBP awaits further investigations, some parallels can be drawn between those of ACBP2 with AtFP6 and with lysoPL2.     

Strigolactones' ability to regulate root development may be executed by induction of the ethylene pathway

The newly defined phytohormones strigolactones (SLs) were recently shown to act as regulators of root development. Their positive effect on root-hair (RH) elongation enabled examination of their cross talk with auxin and ethylene. Analysis of wild-type plants and hormone-signaling mutants combined with hormonal treatments suggested that SLs and ethylene regulate RH elongation via a common regulatory pathway, in which ethylene is epistatic to SLs. The SL and auxin hormonal pathways were suggested to converge for regulation of RH elongation; this convergence was suggested to be mediated via the ethylene pathway, and to include regulation of auxin transport.Key words: strigolactone, auxin, ethylene, root, root hair, lateral rootStrigolactones (SLs) are newly identified phytohormones that act as long-distance shoot-branching inhibitors (reviewed in ref. ¹). In Arabidopsis, SLs have been shown to be regulators of root development and architecture, by modulating primary root elongation and lateral root formation.^2,3 In addition, they were shown to have a positive effect on root-hair (RH) elongation.² All of these effects are mediated via the MAX2 F-box.^2,3In addition to SLs, two other plant hormones, auxin and ethylene, have been shown to affect root development, including lateral root formation and RH elongation.^4–6 Since all three phytohormones (SLs, auxin and ethylene) were shown to have a positive effect on RH elongation, we examined the epistatic relations between them by examining RH length.⁷ Our results led to the conclusion that SLs and ethylene are in the same pathway regulating RH elongation, where ethylene may be epistatic to SLs.⁷ Moreover, auxin signaling was shown to be needed to some extent for the RH response to SLs: the auxin-insensitive mutant tir1-1,⁸ was less sensitive to SLs than the wild type under low SL concentrations.⁷On the one hand, ethylene has been shown to induce the auxin response,^9–12 auxin synthesis in the root apex,^11,12 and acropetal and basipetal auxin transport in the root.^4,13 On the other, ethylene has been shown to be epistatic to SLs in the SL-induced RH-elongation response.⁷ Therefore, it might be that at least for RH elongation, SLs are in direct cross talk with ethylene, whereas the cross talk between SL and auxin pathways may converge through that of ethylene.⁷ The reduced response to SLs in tir1-1 may be derived from its reduced ethylene sensitivity;^7,14 this is in line with the notion of the ethylene pathway being a mediator in the cross talk between the SL and auxin pathways.The suggested ethylene-mediated convergence of auxin and SLs may be extended also to lateral root formation, and may involve regulation of auxin transport. In the root, SLs have been suggested to affect auxin efflux,^3,15 whereas ethylene has been shown to have a positive effect on auxin transport.^4,13 Hence, it might be that in the root, the SLs'' effect on auxin flux is mediated, at least in part, via the ethylene pathway. Ethylene''s ability to increase auxin transport in roots was associated with its negative effect on lateral root formation: ethylene was suggested to enhance polar IAA transport, leading to alterations in the quantity of auxin that unloads into the tissues to drive lateral root formation.⁴ Under conditions of sufficient phosphate, SL''s effect was similar to that of ethylene: SLs reduced the appearance of lateral roots; this was explained by their ability to change auxin flux.³ Taken together, one possibility is that the SLs'' ability to affect auxin flux and thereby lateral root formation in the roots is mediated by induction of ethylene synthesis.To conclude, root development may be regulated by a network of auxin, SL and ethylene cross talk.⁷ The possibility that similar networks exist elsewhere in the SLs'' regulation of plant development, including shoot architecture, cannot be excluded.