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The evolutionary plant-pathogen arms race has equipped plants with the immune system that can defend against pathogens. Pattern-triggered immunity and effector-triggered immunity are two major branches of innate immunity that share immune responses, including oxidative bursts, transcriptional reprogramming, and cell wall modifications such as lignin deposition. In a previous study, we reported that lignin rapidly accumulates in pathogen-infected Arabidopsis leaves and acts as a mechanical barrier, spatially restricting pathogens and cell death. Lignin deposition into the cell wall is a three-step process: monolignol biosynthesis, transport, and polymerization. While monolignol biosynthesis and polymerization are relatively well understood, the mechanism of monolignol transport remains unclear. In this study, we show that macroautophagy/autophagy modulates pathogen-induced lignin formation. Lignification and other immune responses were impaired in autophagy-defective atg (autophagy-related) mutants. In microscopy analyses, monolignols formed punctate structures in response to pathogen infection and colocalized with autophagic vesicles. Furthermore, autophagic activity and lignin accumulation were both enhanced in dnd1 (defense, no death 1) mutant with elevated disease resistance but no cell death and crossing dnd1-1 with atg mutants resulted in a lignin deficit, further supporting that lignin formation requires autophagy. Collectively, these findings demonstrate that lignification, particularly monolignol transport, is achieved through autophagic membrane trafficking in plant immunity.Abbreviations: ABC transporter: ATP-binding cassette transporter; ACD2/AT4G37000: accelerated cell death 2; ATG: autophagy-related; C3′H/AT2G40890: p-coumaroyl shikimate 3-hydroxylase; C4H/AT2G30490: cinnamate 4-hydroxylase; CA: coniferyl alcohol; CaMV: cauliflower mosaic virus; CASP: Casparian strip membrane domain protein; CASPL: CASP-like protein; CBB: Coomassie Brilliant Blue; CCoAOMT1/AT4G34050: caffeoyl-CoA O-methyltransferase 1; CCR1/AT1G15950: cinnamoyl-CoA reductase 1; CFU: colony-forming unit; COMT1/AT5G54160: caffeic acid O-methyltransferase 1; Con A: concanamycin A; DMAC: dimethylaminocoumarin; DND1/AT5G15410: defense, no death 1; CNGC2: cyclic nucleotide-gated channel 2; ER: endoplasmic reticulum; ESB1/AT2G28670/DIR10: enhanced suberin 1; ETI: effector-triggered immunity; EV: extracellular vesicle; F5H/AT4G36220: ferulate-5-hydroxylase; Fluo-3 AM: Fluo-3 acetoxymethyl ester; GFP: green fluorescent protein; HCT/AT5G48930: p-hydroxycinnamoyl-CoA:quinate/shikimate p-hydroxycinnamoyltransferase; HR: hypersensitive response; LAC: laccase; LTG: LysoTracker Green; LSD1/AT4G200380: lesion stimulating disease 1; PAL1/AT2G37040: phenylalanine ammonia-lyase 1; PAMP: pathogen-associated molecular patterns; PCD: programmed cell death; PE: phosphatidylethanolamine; PRX: peroxidase; Pst DC3000: Pseudomonas syringe pv. tomato DC3000; PTI: pattern-triggered immunity; SA: salicylic acid; SD: standard deviation; SID2/AT1G7410: SA induction-deficient 2; UGT: UDP-glucosyltransferase; UPLC: ultraperformance liquid chromatography; UPS: unconventional protein secretion; V-ATPase: vacuolar-type H(+)-translocating ATPase

We previously found that VAMP721/722 SNARE proteins guide secretory vesicles to pathogen-attacking sites during immune responses in Arabidopsis, which suggests that these vesicles should deliver immune molecules. However, the lethality of vamp721 vamp722 double null mutant makes it difficult to understand the nature of cargo transported via VAMP721/722 vesicles. Since VAMP721/722-depleted (VAMP721+/-VAMP722-/- and VAMP721-/-VAMP722+/-) plants show compromised resistance to extracellular pathogens, we assume that an immune protein secreted through the VAMP721/722-engaged exocytosis would be remained more in VAMP721/722-depleted plants than WT. By comparing intracellular proteins between WT and VAMP721/722-depleted plants, we found caffeoyl-CoA O-methyltransferase 1 (CCOAOMT1) involved in the lignin biosynthesis was more abundantly detected in both VAMP721/722-depleted lines than WT. Plants are well-known to deposit secondary cell walls as physical barriers at pathogen-attempting sites. Therefore, extracellular detection of CCOAOMT1 and impaired resistance to Pseudomonas syringae DC3000 in ccoaomt1 plants suggest that plants secrete cell wall-modifying enzymes at least including CCOAOMT1 to reinforce the secondary cell walls for immunity.

Lipid metabolism is associated with colon cancer prognosis and incidence. Stearoyl-CoA desaturase 1 (SCD1), which converts fully saturated fatty acids (SFAs) to monounsaturated fatty acids (MUFAs), has been suggested as a vulnerable target for selective elimination of cancer stem cells (CSCs). However, the clinical significance and physiological role of SCD1 in CSCs has not been well demonstrated. Here, we showed the clinical and biochemical relevance of blocking SCD1 to target CSCs by analyzing human colon cancer data from TCGA and through lipidomic profiling of CSCs with or without SCD1 inhibition using mass spectrometry. Positive associations between SCD1 expression and colorectal cancer patient clinical status and the expression of CSC-related genes (WNT and NOTCH signaling) were found based on TCGA data analysis. Lipidomic profiling of CSCs and bulk cancer cells (BCCs) using mass spectrometry revealed that colon CSCs contained a distinctive lipid profile, with higher free MUFA and lower free SFA levels than in BCCs, suggesting that enhanced SCD1 activity generates MUFAs that may support WNT signaling in CSCs. In addition, all identified phosphatidyl-ethanolamine-containing MUFAs were found at higher levels in CSCs. Interestingly, we observed lower phosphatidyl-serine (18:1/18:0), phosphatidyl-choline (PC; p-18:0/18:1)), and sphingomyelin (SM; d18:1/20:0 or d16:1/22:0) levels in CSCs than in BCCs. Of those, SCD1 inhibition, which efficiently diminished free MUFA levels, increased those specific PC and SM and MUFAs in CSCs promptly. These results suggest that these specific lipid composition is critical for CSC stem cell maintenance. In addition, not only free MUFAs, which are known to be required for WNT signaling, but also other phospholipids, such as SM, which are important for lipid raft formation, may mediate other cell signaling pathways that support CSC maintenance. Comparison of the lipidomic profiles of colon cancer cells with those of previously reported for glioma cells further demonstrated the tissue specific characteristics of lipid metabolism in CSCs.

The gut microbiome has been known to contribute up to ~30% of the energy absorption of the host. Although various beneficial mechanisms of probiotics have been suggested for non-alcoholic fatty liver disease (NAFLD), whether and which probiotics impact the host’s intestinal energy absorption have not yet been quantitatively studied. Here, we suggest a novel mechanism of probiotics against NAFLD, in which Lactobacillus rhamnosus GG, the most common probiotic, shares intestinal fatty acids and prevents the development of diet-induced hepatic steatosis. By using quantitative methods (radioactive tracers and LC–MS) under both in vitro and in vivo conditions, we found that bacteria and hosts competed for fatty acid absorption in the intestine, resulting in decreased weight gain, body fat mass, and hepatic lipid accumulation without differences in calorie intake and excretion in mice fed the probiotic bacteria., Liver disease: Beneficial bacteria divert dietary fats Intestinal bacteria that consume common fatty acids could help protect their hosts against non-alcoholic fatty liver disease (NAFLD). The accumulation of lipids over the course of NAFLD can produce serious inflammation and ultimately lead to liver failure. The gut microbiome plays a prominent role in metabolic health and South Korean researchers led by Hui-Young Lee of Gachon University, Incheon, and Jae-Hak Park of Seoul National University, Seoul, have identified a bacterial species that could help prevent NAFLD. They found that Lactobacillus rhamnosus GG can absorb oleic acid, a fatty acid commonly found in the human diet. Transplantation of these bacteria into high-fat diet-fed mice resulted in weight loss and reduced fat accumulation in the liver. These findings providing a new mechanistic insight into the impact of certain probiotic species on NAFLD.

Cancer stem-like cells (CSLCs) contribute to the initiation and recurrence of tumors and to their resistance to conventional therapies. In this study, small interfering RNA (siRNA)-based screening of ∼4800 druggable genes in 3-dimensional CSLC cultures in comparison to 2-dimensional bulk cultures of U87 glioma cells revealed 3 groups of genes essential for the following: survival of the CSLC population only, bulk-cultured population only, or both populations. While diverse biologic processes were associated with siRNAs reducing the bulk-cultured population, CSLC-eliminating siRNAs were enriched in a few functional categories, such as lipid metabolism, protein metabolism, and gene expression. Interestingly, siRNAs that selectively reduced CSLC only were found to target genes for cholesterol and unsaturated fatty acid synthesis. The lipidomic profile of CSLCs revealed increased levels of monounsaturated lipids. Pharmacologic blockage of these target pathways reduced CSLCs, and this effect was eliminated by addition of downstream metabolite products. The present CSLC-sensitive target categories provide a useful resource that can be exploited for the selective elimination of CSLCs.-Song, M., Lee, H., Nam, M.-H., Jeong, E., Kim, S., Hong, Y., Kim, N., Yim, H. Y., Yoo, Y.-J., Kim, J. S., Kim, J.-S., Cho, Y.-Y., Mills, G. B., Kim, W.-Y., Yoon, S. Loss-of-function screens of druggable targetome against cancer stem-like cells.

The sensitivity of rice to salt stress greatly depends on growth stages, organ types and cultivars. Especially, the roots of young rice seedlings are highly salt-sensitive organs that limit plant growth, even under mild soil salinity conditions. In an attempt to identify metabolic markers of rice roots responding to salt stress, metabolite profiling was performed by H-1-NMR spectroscopy in 38 rice genotypes that varied in biomass accumulation under long-term mild salinity condition. Multivariate statistical analysis showed separation of the control and salt-treated rice roots and rice genotypes with differential growth potential. By quantitative analyses of H-1-NMR data, five conserved salt-responsive metabolic markers of rice roots were identified. Sucrose, allantoin and glutamate accumulated by salt stress, whereas the levels of glutamine and alanine decreased. A positive correlation of metabolite changes with growth potential and salt tolerance of rice genotypes was observed for allantoin and glutamine. Adjustment of nitrogen metabolism in rice roots is likely to be closely related to maintain the growth potential and increase the stress tolerance of rice.

Human papilloma virus (HPV) 16 causes cervical cancer. Induction of oncogenesis by HPV 16 is primarily dependent on the function of E6 and E7 proteins, which inactivate the function of p53 and pRB, respectively. Thus, blocking the activity of the E6 and E7 proteins from HPV 16 is critical to inhibiting oncogenesis during infection. We have expressed and purified soluble HPV 16 E6 and E7 fusion immunoglobulin (Ig), which were combined with the constant region of an Ig heavy chain, in a mammalian system. To assess whether soluble E6 and E7 fusion Igs induce effective cellular immune responses, immature dendritic cells (DCs) were treated with these fusion proteins. Soluble E6 and E7 fusion Igs effectively induced maturation of DCs. Furthermore, immunization with soluble E6 and E7 fusion Igs in mice resulted in antigen-specific activation of T helper 1 (Th1) cells. This is the first comprehensive study to show the molecular basis of how soluble HPV 16 E6 or E7 fusion Igs induces Th1 responses through the maturation of DCs. In addition, we show that DC therapy using soluble HPV E6 and E7 fusion Igs may be a valuable tool for controlling the progress of cervical cancer.

In this study, various solvent systems were applied to obtain a high and consistent recovery rate of low molecular weight plasma proteins (LMPP) from human plasma. A buffer system containing 7 M urea, 2 M thiourea, 25 mM NH(4)HCO(3) + 20% ACN (pH 8.2) produced the highest recovery rate of LMPP. To validate the recovery of cut off membrane (COM) obtained using the urea buffer system, 27 different 30 kDa COMs were used to prepare the LMPP sample which were then subjected to 1-D SDS-PAGE. Statistical analysis showed that the buffer system with COM produced a consistent the recovery of LMPP. In addition, 2-DE analysis was also conducted to determine the relative intensity of each protein spot. When molecular weight ranges over 30 kDa and under 30 kDa were evaluated, 953 and 587 protein spots were observed in the gels, respectively, resulting in a total of 1540 protein spots being resolved. Identification of the major proteins were then performed using a nano-LC/MS system comprised of an HPLC system and an ESI-quadrupole IT MS equipped with a nano-ESI source.

To explore the effects of power frequency magnetic fields (MF) on cell growth in prostate cancer, DU145, PC3, and LNCaP cells were examined in vitro.The cells were exposed to various intensities and durations of 60-Hz sinusoidal MF in combination with various serum concentrations in the media. To analyze MF effects on cell growth, cell counting, trypan blue exclusion assay, Western blot analysis, flow cytometry, enzyme-linked immunosorbent assay (ELISA), semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescence microscopy, and spectrofluorometry were used.MF exposure induced significant cell growth inhibition and apoptosis in an intensity- and time-dependent manner, in which cell cycle arrest, cleaved Caspase-3, and reactive oxygen species (ROS) increased. Pretreatment with a Caspase-3 inhibitor or antioxidant, N-acetyl-L-cysteine (NAC), significantly attenuated MF-induced cell growth inhibition and cell death. Media replacement experiments failed to show any notable change in the MF effects.These results demonstrate 60-Hz sinusoidal MF-activated cell growth inhibition of prostate cancer in vitro. Apoptosis together with cell cycle arrest were the dominant causes of the MF-elicited cell growth inhibition, mediated by MF-induced ROS. These results suggest that a possibility of using 60-Hz MF in radiation therapy of prostate cancer could usefully be investigated.

We used two-dimensional electrophoresis (2-DE) and other proteomic approaches to identify proteins expressed in suspension-cultured rice cells in response to the rice blast fungus, Magnaporthe grisea. Proteins were extracted from suspension-cultured cells at 24 and 48 h after rice blast fungus inoculation or treatment with elicitor or other signal molecules such as jasmonic acid (JA), salicylic acid, and H(2)O(2). The proteins were then polyethylene glycol fractionated before separation by 2-DE. Fourteen protein spots were induced or increased by the treatments, which we analyzed by N-terminal or internal amino acid sequencing. Twelve proteins from six different genes were identified. Rice pathogen-related protein class 10 (OsPR-10), isoflavone reductase like protein, beta-glucosidase, and putative receptor-like protein kinase were among those induced by rice blast fungus; these have not previously been reported in suspension-cultured rice cells. Six isoforms of probenazole-inducible protein (PBZ1) and two isoforms of salt-induced protein (SalT) that responded to blast fungus, elicitor, and JA were also resolved on a 2-DE gel and identified by proteome analysis. The expression level of these induced proteins both in suspension-cultured cells and in leaves of whole plants was analyzed by Western blot. PBZ1, OsPR-10, and SalT proteins from incompatible reactions were induced earlier and to a greater extent than those in compatible reactions. Proteome analysis can thus distinguish differences in the timing and amount of protein expression induced by pathogens and other signal molecules in incompatible and compatible interactions.

As an initial step to the comprehensive proteomic analysis of Panax ginseng C. A. Meyer, protein mixtures extracted from the cultured hairy root of Panax ginseng were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). The protein spots were analyzed and identified by peptide finger printing and internal amino acid sequencing by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and electrospray ionization quadrupole-time of flight mass spectrometry (ESI Q-TOF MS), respectively. More than 300 protein spots were detected on silver stained two-dimensional (2-D) gels using pH 3-10, 4-7, and 4.5-5.5 gradients. Major protein spots (159) were analyzed by peptide fingerprinting or de novo sequencing and the functions of 91 of these proteins were identified. Protein identification was achieved using the expressed sequence tag (EST) database from Panax ginseng and the protein database of plants like Arabidopsis thaliana and Oryza sativa. However, peptide mass fingerprinting by MALDI-TOF MS alone was insufficient for protein identification because of the lack of a genome database for Panax ginseng. Only 17 of the 159 protein spots were verified by peptide mass fingerprinting using MALDI-TOF MS whereas 87 out of 102 protein spots, which included 13 of the 17 proteins identified by MALDI-TOF MS, were identified by internal amino acid sequencing using tandem mass spectrometry analysis by ESI Q-TOF MS. When the internal amino acid sequences were used as identification markers, the identification rate exceeded 85.3%, suggesting that a combination of internal sequencing and EST data analysis was an efficient identification method for proteome analysis of plants having incomplete genome data like ginseng. The 2-D patterns of the main root and leaves of Panax ginseng differed from that of the cultured hairy root, suggesting that some proteins are exclusively expressed by different tissues for specific cellular functions. Proteome analysis will undoubtedly be helpful for understanding the physiology of Panax ginseng.

From hemocytes of the tunicate Halocynthia aurantium we purified a new antimicrobial peptide named halocidin. The native peptide had a mass of 3443 Da and comprised two different subunits containing 18 amino acid residues (WLNALLHHGLNCAKGVLA) and 15 residues (ALLHHGLNCAKGVLA), which were linked covalently by a single cystine disulfide bond. Two different monomers were separately synthesized and used to make three additional isoforms (15 residue homodimer, 18 residue homodimer, heterodimer). In antimicrobial assays performed with synthetic peptides of halocidin, it was confirmed that congeners of the 18 residue monomer were more active than those of the 15 residue monomer against methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa.

It was previously reported that the amounts of lysophosphatidylcholines (lysoPCs), which are naturally occurring bioactive lipid molecules, significantly increase following pathogen inoculation, as determined using ultraperformance liquid chromatography-quadrupole-time of flight/mass spectrometry analyses. Here, real-time quantitative RT-PCR was performed for the phospholipase A2 (PLA2) genes, Nt1PLA2 and Nt2PLA2, which are responsible for LysoPCs generation. The transcription level of Nt2PLA2 in pathogen-infected tobacco plants transiently peaked at 1h and 36 h, whereas induction of Nt1PLA2 transcription peaked at 36 h. A prominent biphasic ROS accumulation in lysoPC (C18:1(9Z))-treated tobacco leaves was also observed. Transcription of NtRbohD, a gene member of NADPH oxidase, showed biphasic kinetics upon lysoPC 18:1 treatment, as evidenced by an early transient peak in phase I at 1h and a massive peak in phase II at 12h. Each increase in NtACS2 and NtACS4 transcription, gene members of the ACC synthase family, was followed by biphasic peaks of ethylene production after lysoPC 18:1 treatment. This suggested that lysoPC (C18:1)-induced ethylene production was regulated at the transcriptional level of time-dependent gene members. LysoPC 18:1 treatment also rapidly induced cell damage. LysoPC 18:1-induced cell death was almost completely abrogated in ROS generation-impaired transgenic plants (rbohD-as and rbohF-as), ethylene production-impaired transgenic plants (CAS-AS and CAO-AS), and ethylene signaling-impaired transgenic plants (Ein3-AS), respectively. Taken together, pathogen-induced lysoPCs enhance pathogen susceptibility accompanied by ROS and ethylene biosynthesis, resulting in chlorophyll degradation and cell death. Expression of PR genes (PR1-a, PR-3, and PR-4b) and LOX3 was strongly induced in lysoPC 18:1-treated leaves, indicating the involvement of lysoPC 18:1 in the defense response. However, lysoPC 18:1 treatment eventually resulted in cell death, as evidenced by metacaspase gene expression. Therefore, a hypothesis is proposed that the antipathogenic potential of lysoPC 18:1 is dependent on how quickly it is removed from cells for avoidance of lysoPC toxicity.

Background To investigate the molecular and cellular pathogenesis underlying myocarditis, we used an experimental autoimmune myocarditis (EAM)-induced heart failure rat model that represents T cell mediated postinflammatory heart disorders. Results By performing unbiased 2-dimensional electrophoresis of protein extracts from control rat heart tissues and EAM rat heart tissues, followed by nano-HPLC-ESI-QIT-MS, 67 proteins were identified from 71 spots that exhibited significantly altered expression levels. The majority of up-regulated proteins were confidently associated with unfolded protein responses (UPR), while the majority of down-regulated proteins were involved with the generation of precursor metabolites and energy metabolism in mitochondria. Although there was no difference in AKT signaling between EAM rat heart tissues and control rat heart tissues, the amounts and activities of extracellular signal-regulated kinase (ERK)-1/2 and ribosomal protein S6 (rpS6) were significantly increased. By comparing our data with the previously reported myocardial proteome of the Coxsackie viruses of group B (CVB)-mediated myocarditis model, we found that UPR-related proteins were commonly up-regulated in two murine myocarditis models. Even though only two out of 29 down-regulated proteins in EAM rat heart tissues were also dysregulated in CVB-infected rat heart tissues, other proteins known to be involved with the generation of precursor metabolites and energy metabolism in mitochondria were also dysregulated in CVB-mediated myocarditis rat heart tissues, suggesting that impairment of mitochondrial functions may be a common underlying mechanism of the two murine myocarditis models. Conclusions UPR, ERK-1/2 and S6RP signaling were activated in both EAM- and CVB-induced myocarditis murine models. Thus, the conserved components of signaling pathways in two murine models of acute myocarditis could be targets for developing new therapeutic drugs or methods aimed at treating enigmatic myocarditis.

Pine wilt disease (PWD) is one of the most devastating forest diseases in Asia and Europe. The pine wood nematode, Bursaphelenchus xylophilus, has been identified as the pathogen underlying PWD, although the pathology is not completely understood. At present, diagnosis and confirmation of PWD are time consuming tasks that require nematode extraction and microscopic examination. To develop a more efficient detection method for B. xylophilus, we first generated monoclonal antibodies (MAbs) specific to B. xylophilus. Among 2304 hybridoma fusions screened, a hybridoma clone named 3-2A7-2H5 recognized a single protein from B. xylophilus specifically, but not those from other closely related nematodes. We finally selected the MAb clone 3-2A7-2H5-D9-F10 (D9-F10) for further studies. To identify the antigenic target of MAb-D9-F10, we analyzed proteins in spots, fractions, or bands isolated from SDS-PAGE, two-dimensional electrophoresis, anion exchange chromatography, and immunoprecipitation via nano liquid chromatography electrospray ionization quadrupole ion trap mass spectrometry (nano-LC-ESI-Q-IT-MS). Peptides of galactose-binding lectin-1 of B. xylophilus (Bx-LEC-1) were commonly detected in several proteomic analyses, demonstrating that this LEC-1 is the antigenic target of MAb-D9-F10. The localization of MAb-D9-F10 immunoreactivities at the area of the median bulb and esophageal glands suggested that the Bx-LEC-1 may be involved in food perception and digestion. The Bx-LEC-1 has two nonidentical galactose-binding lectin domains important for carbohydrate binding. The affinity of the Bx-LEC-1 to d-(+)-raffinose and N-acetyllactosamine were much higher than that to l-(+)-rhamnose. Based on this combination of evidences, MAb-D9-F10 is the first identified molecular biomarker specific to the Bx-LEC-1.

OREB1 is a rice ABRE binding factor characterized by the presence of multiple highly-conserved phosphorylation domains (C1, C2, C3, and C4) and two kinase recognition motifs, RXXS/T and S/TXXE/D, within different functional domains. An in vitro kinase assay showed that OREB1 is phosphorylated not only by the SnRK2 kinase, but also by other Ser/Thr protein kinases, such as CaMKII, CKII, and SnRK3. Furthermore, the N-terminal phosphorylation domain C1 was found to be differentially phosphorylated by the SnRK2/SnRK3 kinase and by hyperosmotic/cold stress, suggesting that the C1 domain may function in decoding different signals. The phosphorylation-mediated regulation of OREB1 activity was investigated through mutation of the SnRK2 recognition motif RXXS/T within each phosphorylation module. OREB1 contains a crucial nine-amino acid transactivation domain located near the phosphorylation module C1. Deletion of the C1 domain increased OREB1 activity, whereas mutation of Ser 44, Ser 45, and Ser 48 of the C1 domain to aspartates decreased OREB1 activity. In the C2 domain, a double mutation of Ser 118 and Ser 120 to alanines suppressed OREB1 activity. These findings strongly suggest that selective phosphorylation of the C1 or C2 modules may positively or negatively regulate OREB1 transactivation. In addition, mutation of Ser 385 of the C4 domain to alanines completely abolished the interaction between OREB1 and a rice 14-3-3 protein, GF14d, suggesting that SnRK2-mediated phosphorylation may regulate this interaction. These results indicate that phosphorylation domains of OREB1 are not functionally redundant and regulate at least three different functions, including transactivation activity, DNA binding, and protein interactions. The multisite phosphorylation of OREB1 is likely a key for the fine control of its activity and signal integration in the complex stress signaling network of plant cells.

Psychrophilicity of Gram-positive bacterium, Bacillus psychrosaccharolyticus was investigated in a proteomic approach. One hundred and thirty-one protein spots were analyzed by electrospray ionization-quadrupole-time of flight-tandem mass spectrometry and identified using an unpublished translated contig database as well as a nonredundant Gram-positive bacteria protein database from NCBI because of the lack of a genome sequence of this organism. Results focused on proteomic behavior of cold-response show that global up-regulation of metabolic functions and protective mechanism by stress responses might play a major role in psychrophilicity of B. psychrosaccharolyticus.

We performed comparative proteomic analyses in order to understand the physiological responses of ginseng (Panax ginseng C. A. Meyer) to high light (HL). As a first step, we analyzed the proteins expressed in ginseng leaves. Proteins extracted from leaves were separated by two-dimensional polyacrylamide gel electrophoresis. Protein spots were identified by tandem mass spectra analysis using electrospray ionization quadrupole-time of flight mass spectrometry (ESI Q-TOF MS). We used a ginseng expressed sequence tag (EST) database as well as a nonredundant protein database from NCBI to identify proteins. Eighty-one proteins were identified using the nr protein database, 51 of which were also verified from the ginseng EST database. An additional 66 proteins were identified only from the ginseng EST database. Proteins that function in energy metabolism, protein stabilization, and protection against oxidative stress were abundant. To understand the light responses of ginseng leaves, we studied time dependent changes in expressed proteins produced by 0-4 h of HL exposure. Six HL-responsive proteins were identified: three proteins were up-regulated (cytosolic small heat-shock protein, cytosolic ascorbate peroxidase, and putative major latex-like protein) and three proteins were down-regulated (Rieske Fe/S protein, putative 3-beta hydroxysteroid dehydrogenase/isomerase-like protein, and oxygen-evolving enhancer-like protein). Our results show that the ginseng EST database combined with ESI Q-TOF MS analysis can be used to identify ginseng proteins and to elucidate the protective mechanism of ginseng against HL induced damage.

Acinetobacter lwoffii K24 is a soil bacterium that can use aniline as a sole carbon and nitrogen source (by beta-ketoadipate pathway genes (cat genes)) and has two copies of catABC gene separately located on the chromosome. In order to identify aniline-induced proteins, two-dimensional electrophoresis (2-DE) was applied to soluble protein fractions of A. lwoffii K24 cultured in aniline and succinate media. In the range of pH3-10, more than 370 spots were detected on the silver stained gels. Interestingly, more than 20 spots were selectively induced on aniline-cultured bacteria. Twenty-three protein spots of A. lwoffii K24 were analyzed by N-terminal microsequencing and internal microsequencing with in-gel digestion. Of 20 aniline induced protein spots, we identified six beta-ketoadipate pathway genes, one subunit of amino group transfer (putative subunit of aniline oxygenase), malate dehydrogenase, putative ABC transporter, putative hydrolase, HHDD isomerase, and five unknown proteins. Especially in case of two catechol 1,2-dioxygenases (CDI1 and CDI2), more than three isotypes were detected on the 2D gel. This study showed that the proteome analysis of A. lwoffii K24 may be helpful for identification of genes induced by aniline and understanding of their function in the cell.

Background The rice roots are highly salt-sensitive organ and primary root growth is rapidly suppressed by salt stress. Sucrose nonfermenting 1-related protein kinase2 (SnRK2) family is one of the key regulator of hyper-osmotic stress signalling in various plant cells. To understand early salt response of rice roots and identify SnRK2 signaling components, proteome changes of transgenic rice roots over-expressing OSRK1, a rice SnRK2 kinase were investigated. Results Proteomes were analyzed by two-dimensional electrophoresis and protein spots were identified by LC-MS/MS from wild type and OSRK1 transgenic rice roots exposed to 150 mM NaCl for either 3 h or 7 h. Fifty two early salt -responsive protein spots were identified from wild type rice roots. The major up-regulated proteins were enzymes related to energy regulation, amino acid metabolism, methylglyoxal detoxification, redox regulation and protein turnover. It is noted that enzymes known to be involved in GA-induced root growth such as fructose bisphosphate aldolase and methylmalonate semialdehyde dehydrogenase were clearly down-regulated. In contrast to wild type rice roots, only a few proteins were changed by salt stress in OSRK1 transgenic rice roots. A comparative quantitative analysis of the proteome level indicated that forty three early salt-responsive proteins were magnified in transgenic rice roots at unstressed condition. These proteins contain single or multiple potential SnRK2 recognition motives. In vitro kinase assay revealed that one of the identified proteome, calreticulin is a good substrate of OSRK1. Conclusions Our present data implicate that rice roots rapidly changed broad spectrum of energy metabolism upon challenging salt stress, and suppression of GA signaling by salt stress may be responsible for the rapid arrest of root growth and development. The broad spectrum of functional categories of proteins affected by over-expression of OSRK1 indicates that OSRK1 is an upstream regulator of stress signaling in rice roots. Enzymes involved in glycolysis, branched amino acid catabolism, dnaK-type molecular chaperone, calcium binding protein, Sal T and glyoxalase are potential targets of OSRK1 in rice roots under salt stress that need to be further investigated.