Superoxide dismutase, peroxidase, and germin-like protein activity in plasma membranes and apoplast of maize roots

Summary. The analysis of plasma membranes from maize roots by native gel electrophoresis revealed the existence of Mn-containing 120 kDa and CuZn-containing 70, 40, and 15 kDa superoxide dismutase (SOD) isoform activities. Isoelectric focusing of the plasma membranes differentiated anionic SOD isoforms with a pI of about 5 and cationic SOD isoforms at pI 8.6. Solubilization of the plasma membrane proteins further separated the cationic SOD into pI 8.6, 8.2, 8.4, and 7.2 isoforms. Double staining for both SOD and peroxidase activities showed an overlap of these activities only in the case of the high-molecular-mass (ca. 120 kDa) isoforms. High-temperature treatments demonstrated that the 120 kDa isoform was active even at 100 °C, indicating that it was a germin-like protein with superoxide-dismutating activity, different from the peroxidase with a similar molecular mass and the lower-molecular-mass CuZn-containing superoxide dismutases. These results are compared to those obtained from whole-tissue extract and apoplastic fluid. Correspondence and reprints: Maize Research Institute, POB 89-Zemun, 11081 Belgrade, Serbia and Montenegro.     

Transgenically expressed rice germin-like protein1 in tobacco causes hyper-accumulation of H2O2 and reinforcement of the cell wall components

Our recent report documented that the rice germin-like protein1 (OsGLP1), being a cell wall-associated protein involves in disease resistance in rice and possesses superoxide dismutase (SOD) activity as recognized by heterologous expression in tobacco. In the present study, the transgenic tobacco plants were analyzed further to decipher the detailed physiological and biochemical functions of the OsGLP1 and its associated SOD activity. The transgenic tobacco lines expressing SOD-active OsGLP1 showed tolerance against biotic and abiotic stresses mitigated by hyper-accumulating H₂O₂ upon infection by fungal pathogen (Fusarium solani) and treatment to chemical oxidizing agent (ammonium persulfate), respectively. Histological staining revealed enhanced cross-linking of the cell wall components in the stem tissues of the transgenic plants. Fourier transform infrared spectroscopy (FTIR) analysis of the biopolymer from the stem tissues of the transgenic and untransformed plants revealed differential banding pattern of the spectra corresponding to various functional groups. Our findings demonstrate that the OsGLP1 with its inherent SOD activity is responsible for hyper-accumulation of H₂O₂ and reinforcement of the cell wall components.     

Cotton-fiber germin-like protein. II: Immunolocalization,purification, and functional analysis

Cotton (Gossypium hirsutum L.) contains a germin-like protein (GLP), GhGLP1, that shows tissue-specific accumulation in fiber. The fiber GLP is an oligomeric, glycosylated protein with a subunit size of approximately 25.5 kDa. Accumulation of GhGLP1 occurs during the period of fiber elongation [4–14 days post-anthesis (DPA)]. During early phases of fiber development (2–4 DPA), GhGLP1 localizes to cytoplasmic vesicles as shown by confocal immunofluorescent microscopy. In slightly older fibers (7–10 DPA), GhGLP1 localizes to the apoplast. In other plants, germins and GLPs have been reported to have enzymatic activities including oxalate oxidase (OxO), superoxide dismutase, and ADP-glucose pyrophosphatase. Cotton fiber extracts did not contain OxO activity, nor did intact fibers stain for OxO activity. A four-step purification protocol involving ammonium sulfate precipitation of a 1.0 M NaCl extract, ion-exchange chromatography on DEAE-Trisacryl M, lectin-affinity chromatography, and gel filtration chromatography resulted in electrophoretically pure GhGLP1. While 1.0 M NaCl extracts from 10–14 DPA fiber contained superoxide dismutase and phosphodiesterase activities, GhGLP1 could be separated from both enzyme activities by the purification protocol. Although a GLP accumulates in the cotton fiber apoplast during cell elongation, the function of this protein in fiber growth and development remains unknown.Abbreviations ABP Auxin binding protein - AGPPase ADP-Glucose pyrophosphatase/phosphodiesterase - bis-PNPP Bis-p-nitrophenol phosphate - ConA Concanavalin A - DOA Day of anthesis - DPA Days post-anthesis - GLP Germin-like protein - Mn-SOD Manganese superoxide dismutase - OxO Oxalate oxidase - PBS Phosphate-buffered saline     

Apoptosis signal-regulating kinase-1 aggravates ROS-mediated striatal degeneration in 3-nitropropionic acid-infused mice

Apoptosis signal-regulating kinase-1 (ASK1), an early signaling element in the cell death pathway, has been suggested to participate in the pathology of neurodegenerative diseases, which may be associated with environmental factors that impact the diseases. Although it is not entirely elucidated, 3-nitropropionic acid (3-NP) provokes mitochondrial dysfunction and selectively forms striatal lesions similar to those found in Huntington’s disease. The current study investigated whether ASK1 is involved in striatal pathology following chronic systemic infusion of 3-NP. The results show that ASK1 acts as a primary mediator of there active oxygen species (ROS) cell death signal cascade in the 3-NP-damaged striatal region by disrupting the positive feedback cycle. In 3-NP-infused striatal lesions, ROS increased ASK1. Superoxide dismutase transgenic (SOD-tg) mice reduced ASK1by scavenging ROS, and reduction of ASK1leads to a reduction in cell death. However, ASK1 down-regulation in 3-NP infusion mice also decreased striatal cell death without scavenging ROS. In contrast decreasing cell death by si-ASK1 treatment along with 3-NP in both SOD tg and wild-type mice (wt), cell death rebounded when ASK1 peptide was added to SOD tg mice. The present study suggests that ROS-inducing ASK1 may be an important step in the pathogenesis of 3-NP infused striatal lesions in murine brains.     

Functional characterization of germin and germin-like protein genes in various plant species using transgenic approaches

The important role of germins (GER) and genes coding for germin-like proteins (GLP) in responses against various stresses in both homologous and heterologous systems is well validated. This review summarizes the work on their functional validation using various biotechnological approaches. The genes are widely expressed during a specific period of plant growth and development, and exhibit a pattern of evolutionary subfunctionalization at both the intracellular and whole plant level. Their applications against various biotic and abiotic stresses, especially against fungal pathogens, are enormous. Although the validation of these proteins against various stresses has led to the development of commercially and agronomically important transgenic plants, much work is still needed to exploit this ever-expanding repertoire of genes and deploy them for commercial use. Historical progress of genetic engineering in GERs and GLPs is reviewed, and future prospects for their potential role in crop improvement are highlighted.     

Death-associated protein kinase 1 has a critical role in aberrant tau protein regulation and function

The presence of tangles composed of phosphorylated tau is one of the neuropathological hallmarks of Alzheimer''s disease (AD). Tau, a microtubule (MT)-associated protein, accumulates in AD potentially as a result of posttranslational modifications, such as hyperphosphorylation and conformational changes. However, it has not been fully understood how tau accumulation and phosphorylation are deregulated. In the present study, we identified a novel role of death-associated protein kinase 1 (DAPK1) in the regulation of the tau protein. We found that hippocampal DAPK1 expression is markedly increased in the brains of AD patients compared with age-matched normal subjects. DAPK1 overexpression increased tau protein stability and phosphorylation at multiple AD-related sites. In contrast, inhibition of DAPK1 by overexpression of a DAPK1 kinase-deficient mutant or by genetic knockout significantly decreased tau protein stability and abolished its phosphorylation in cell cultures and in mice. Mechanistically, DAPK1-enhanced tau protein stability was mediated by Ser71 phosphorylation of Pin1, a prolyl isomerase known to regulate tau protein stability, phosphorylation, and tau-related pathologies. In addition, inhibition of DAPK1 kinase activity significantly increased the assembly of MTs and accelerated nerve growth factor-mediated neurite outgrowth. Given that DAPK1 has been genetically linked to late onset AD, these results suggest that DAPK1 is a novel regulator of tau protein abundance, and that DAPK1 upregulation might contribute to tau-related pathologies in AD. Therefore, we offer that DAPK1 might be a novel therapeutic target for treating human AD and other tau-related pathologies.     

Germin-like genes are expressed during somatic embryogenesis and early development of conifers

Germins and germin-like proteins (GLPs) are members of a superfamily of proteins widely distributed in plants. Their localization within the extracellular matrix and in some cases their hydrogen peroxide-producing activity suggests that these proteins are involved in cell wall metabolism during stress responses and developmental processes. Several very highly conserved conifer GLPs have been identified in somatic embryo tissues. In order to gain more knowledge on their potential involvement in the development of this particular tissue, we have characterized a new GLP gene, LmGER1 in hybrid larch. Anti-GLP immunserum and in-gel activity analyses suggested the presence of superoxide dismutase activity in apoplastic proteins from larch somatic embryos. These results could indicate a possible role for LmGER1 in this physiological process. The expression of LmGER1 has been followed during the maturation of somatic embryos and in different organs of young plantlets by homologous transformation with a promoter-gus construct. This promoter was activated in the root cap of young embryos and, later on, in the cotyledons and in the vascular procambium and xylem. Furthermore, the importance of this gene in embryo development was evaluated by transforming embryonal masses with a gene construct encoding a hairpin RNA leading to gene silencing. The potential role of LmGER1 in cross-linking of cell wall components is discussed.     

TaCOLD1 defines a new regulator of plant height in bread wheat

Plant height is among the most important agronomic traits that influence crop yield. However, in addition to the Rht‐1 alleles, the molecular basis of plant height in bread wheat remains largely unclear. Based on wheat gene expression profiling analysis, we identify a light‐regulated gene from bread wheat, designated as TaCOLD1, whose encoding protein is homologous to cold sensor COLD1 in rice. We show that TaCOLD1 protein is localized to the endoplasmic reticulum (ER) and plasma membrane. Phenotypic analyses show that overexpression of a mutated form of TaCOLD1 (M187K) in bread wheat cultivar Kenong199 (Rht‐B1b) background resulted in an obvious reduction in plant height. Further, we demonstrate that the hydrophilic loop of TaCOLD1 (residues 178–296) can interact with TaGα‐7A (the α subunit of heterotrimeric G protein) protein but not TaGα‐1B, and the mutation (M187K) in TaCOLD1 remarkably enhances its interaction with TaGα‐7A. Physical interaction analyses show that the C‐terminal region of TaGα‐7A, which is lacking in the TaGα‐1B protein, is necessary for its interaction with TaCOLD1. Intriguingly, the C‐terminal region of TaGα‐7A is also physically associated with the TaDEP1 protein (an atypical Gγ subunit). Significantly, we discover that TaCOLD1 and mTaCOLD1 (M187K) can interfere with the physical association between TaGα‐7A and TaDEP1. Together, this study reveals that TaCOLD1 acts as a novel regulator of plant height through interfering with the formation of heterotrimeric G protein complex in bread wheat and is a valuable target for the engineering of wheat plant architecture.     

Arabidopsis thaliana germin-like proteins: common and specific features point to a variety of functions

 Germin-like proteins (GLPs) are ubiquitous plant proteins encoded by diverse multigene families. It is not known whether they share germin's unusual biochemical properties and oxalate oxidase activity. Using specific antibodies, we have studied three GLPs (AtGER1, AtGER2 and AtGER3) in Arabidopsis thaliana (L.) Heynh. as well as in transgenic tobacco (Nicotiana tabacum L.) plants overexpressing these proteins. Like wheat (Triticum aestivum L.) germin, these Arabidopsis GLPs are associated with the extracellular matrix (ECM) and they also seem to exist as two glycosylated isoforms. However, none of them is an oxalate oxidase. Although GLPs display several conserved features, each has its specific characteristics. Both AtGER2 and AtGER3 are oligomeric proteins that share germin's resistance to pepsin and to dissociation by heat and SDS. In contrast, AtGER1 seems to exist as a monomer. The GLPs may interact with the ECM in a variety of ways, since each is efficiently extracted by different conditions. In addition, germins and GLPs all bind Cibacron Blue, a dye often but not exclusively used for the purification of enzymes having nucleotide cofactors. In the case of AtGER2, binding to the dye is so tight that it almost allows a one-step purification of this protein. The variety of sequences, expression patterns and biochemical features indicates that GLPs could be a class of receptors localized in the ECM and involved in physiological and developmental processes as well as stress response. Received: 28 June 1999 / Accepted: 6 December 1999     

Prion protein participates in the regulation of classical and alternative activation of BV2 microglia

The cellular prion protein (PrP^C) is a glycoprotein anchored by glycosylphosphatidylinositol (GPI) to the cell surface and is abundantly expressed in the central nervous system. Numerous studies have suggested a protective function for PrP^C, including protection from ischemic and excitotoxic lesions and several apoptotic insults, and recent reports have shown that PrP^C has a context‐dependent neuroprotective function. In this study, we investigated the effect of PPNP down‐regulation on various forms of microglial activation. We first examined the mRNA expression of PRNP upon exposure to IFN‐γ, IL‐4, or IL‐10 in BV2 microglia. We then analyzed the effect of si‐RNA‐mediated disruption of PRNP on different parameters of microglial activation in IFN‐γ‐, IL‐4‐, or IL‐10‐stimulated microglia. The results showed that PRNP mRNA expression was invariably down‐regulated in microglia upon exposure to IFN‐γ, IL‐4, or IL‐10. PRNP silencing prior to cytokines treatment reduced the responsiveness of microglia to INF‐γ treatment, significantly altered IL‐4‐induced microglial activation phenotype, and had no effect on IL‐10‐induced microglial activation. Together, these results support a role of PrP^C in the modulation of the shift of microglia from a quiescent state to an activated phenotype and in the regulation of the microglial response during classical and alternative activation.     

The role of kupffer cell oxidant production in early ethanol-induced liver disease

Considerable evidence for a role of Kupffer cells in alcoholic liver disease has accumulated and they have recently been shown to be a predominant source of free radicals. Several approaches including pharmacological agents, knockout mice, and viral gene transfer have been used to fill critical gaps in understanding key mechanisms by which Kupffer cell activation, oxidant formation, and cytokine production lead to liver damage and subsequent pathogenesis. This review highlights new data in support of the hypothesis that Kupffer cells play a pivotal role in hepatotoxicity due to ethanol by producing oxidants via NADPH oxidase.     

Functional analysis of rice NPR1-like genes reveals that OsNPR1/NH1 is the rice orthologue conferring disease resistance with enhanced herbivore susceptibility

The key regulator of salicylic acid (SA)-mediated resistance, NPR1, is functionally conserved in diverse plant species, including rice (Oryza sativa L.). Investigation in depth is needed to provide an understanding of NPR1-mediated resistance and a practical strategy for the improvement of disease resistance in the model crop rice. The rice genome contains five NPR1-like genes. In our study, three rice homologous genes, OsNPR1/NH1, OsNPR2/NH2 and OsNPR3, were found to be induced by rice bacterial blight Xanthomonas oryzae pv. oryzae and rice blast Magnaporthe grisea, and the defence molecules benzothiadiazole, methyl jasmonate and ethylene. We confirmed that OsNPR1 is the rice orthologue by complementing the Arabidopsis npr1 mutant. Over-expression of OsNPR1 conferred disease resistance to bacterial blight, but also enhanced herbivore susceptibility in transgenic plants. The OsNPR1-green fluorescent protein (GFP) fusion protein was localized in the cytoplasm and moved into the nucleus after redox change. Mutations in its conserved cysteine residues led to the constitutive localization of OsNPR1(2CA)-GFP in the nucleus and also abolished herbivore hypersensitivity in transgenic rice. Different subcellular localizations of OsNPR1 antagonistically regulated SA- and jasmonic acid (JA)-responsive genes, but not SA and JA levels, indicating that OsNPR1 might mediate antagonistic cross-talk between the SA- and JA-dependent pathways in rice. This study demonstrates that rice has evolved an SA-mediated systemic acquired resistance similar to that in Arabidopsis, and also provides a practical approach for the improvement of disease resistance without the penalty of decreased herbivore resistance in rice.     

AtMTM1, a novel mitochondrial protein,may be involved in activation of the manganese-containing superoxide dismutase in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Mtm1p is essential for the posttranslational activation of manganese-containing superoxide dismutase (SOD2) in Saccharomyces cerevisiae; however, whether the same holds true for Arabidopsis thaliana is unknown. In this study, by using the yeast mtm1 mutant complementation method, we identified a putative MTM gene (AtMTM1, At4g27940) that is necessary for SOD2 activation. Further, analysis of SOD activity revealed that an SOD2 defect is rescued in the yeast mutant Y07288 harboring the AtMTM1 gene. Related mRNA-level analysis showed the AtMTM1 gene is induced by paraquat but not by hydrogen peroxide, which indicates that this gene is related to the superoxide scavenger SOD. In addition, an AtMTM1::GFP fusion construct was transiently expressed in the protoplasts, and it was localized to the mitochondria. Furthermore, sequence deletion analysis of AtMTM1 revealed that the code region (amino acid (aa) 60–198) of Mtm1p plays an important role in localization of the protein to the mitochondria. Regulation of AtMTM1 gene expression was analyzed using a fusion construct of the 1,766 bp AtMTM1 promoter and the GUS (β-glucuronidase) reporter gene. The screen identified GUS reporter gene expression in the developing cotyledons, leaves, roots, stems, and flowers but not in the siliques. Our results suggest that AtMTM1 encodes a mitochondrial protein that may be playing an important role in activation of MnSOD1 in Arabidopsis.     

Mutant SOD1 linked to familial amyotrophic lateral sclerosis,but not wild-type SOD1, induces ER stress in COS7 cells and transgenic mice

Mutations in a Cu, Zn-superoxide dismutase (SOD1) cause motor neuron death in human familial amyotrophic lateral sclerosis (FALS) and its mouse model, suggesting that mutant SOD1 has a toxic effect on motor neurons. However, the question of how the toxic function is gained has not been answered. Here, we report that the mutant SOD1s linked to FALS, but not wild-type SOD1, aggregated in association with the endoplasmic reticulum (ER) and induced ER stress in the cDNA-transfected COS7 cells. These cells showed an aberrant intracellular localization of mitochondria and microtubules, which might lead to a functional disturbance of the cells. Motor neurons of the spinal cord in transgenic mice with a FALS-linked mutant SOD1 also showed a marked increase of GRP78/BiP, an ER-resident chaperone, just before the onset of motor symptoms. These data suggest that ER stress is involved in the pathogenesis of FALS with an SOD1 mutation.     

泛素化在植物抗病中的作用

泛素化作为植物体内一种广泛存在的调控细胞反应的机制,参与调控植物抗病反应。本文综述了泛素化系统在植物抗病反应中的功能及作用机制,重点介绍了CRLs型E3泛素连接酶和RING/U-box型E3泛素连接酶如何参与调控植物抗病信号途径,以及病原物通过效应蛋白和毒性因子调控植物抗病性的分子机理,为阐明植物抗病机理和植物病害防治方法提供参考。