GSH threshold requirement for NO-mediated expression of the Arabidopsis AtFer1 ferritin gene in response to iron

Iron treatment of Arabidopsis cultured cells promotes a rapid NO burst within chloroplasts, necessary for up-regulation of the AtFer1 ferritin gene expression. The same occurs in Arabidopsis leaf chloroplasts, and is dependent upon the GSH content of plants. A leaf GSH concentration threshold between 10 and 50 nmol GSHg(-1) FW is required for full induction of AtFer1 gene expression in response to iron.     

Expression of a rice gene OsNOA1 re-establishes nitric oxide synthesis and stress-related gene expression for salt tolerance in Arabidopsis nitric oxide-associated 1 mutant Atnoa1

Nitric oxide (NO) has been indicated in regulating a wide-spectrum of plant developmental events and stress responses. An Arabidopsis gene AtNOA1 encodes a NO-associated protein, which plays a role in salt tolerance. We employed the knockout mutant for AtNOA1, Atnoa1 that is sensitive to salinity, as a tool to evaluate the functions of a rice homologous gene, OsNOA1.OsNOA1 transgenic expression rescued Atnoa1 in seedling development and vegetative growth under normal conditions, enhanced the salt tolerance of Atnoa1 in seed germination, seedling root growth and chlorophyll synthesis, and reduced Na⁺/K⁺ ratio in Atnoa1; NO relative content assay implicates that NO synthesis was re-established via OsNOA1 expression in Atnoa1; Northern blot and Semi-Q RT-PCR assays demonstrate that salt tolerance-related gene expression was re-established as well via OsNOA1 expression in Atnoa1.Our data indicate that the re-establishment of NO synthesis and salt tolerance-related gene expression by OsNOA1 expression may account for the restoration of Atnoa1 in terms of developmental and salt tolerance phenotypes. All the above results point to a notion that OsNOA1 may play similar roles as AtNOA1, and NO involvement in salt tolerance may be ascribed to its regulation of salt tolerance-related gene expression.     

A nitric oxide burst precedes apoptosis in angiosperm and gymnosperm callus cells and foliar tissues

Leaves and callus of Kalancho? daigremontiana and Taxus brevifolia were used to investigate nitric oxide-induced apoptosis in plant cells. The effect of nitric oxide (NO) was studied by using a NO donor, sodium nitroprusside (SNP), a nitric oxide-synthase (NOS) inhibitor, N:(G)-monomethyl-L-arginine (NMMA), and centrifugation (an apoptosis-inducing treatment in these species). NO production was visualized in cells and tissues with a specific probe, diaminofluorescein diacetate (DAF-2 DA). DNA fragmentation was detected in situ by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) method. In both species, NO was detected diffused in the cytosol of epidermal cells and in chloroplasts of guard cells and leaf parenchyma cells. Centrifugation increased NO production, DNA fragmentation and subsequent cell death by apoptosis. SNP mimicked centrifugation results. NMMA significantly decreased NO production and apoptosis in both species. The inhibitory effect of NMMA on NO production suggests that a putative NOS is present in Kalancho? and Taxus cells. The present results demonstrated the involvement of NO on DNA damage leading to cell death, and point to a potential role of NO as a signal molecule in these plants.     

Glutathione depletion inhibits IL-1 beta-stimulated nitric oxide production by reducing inducible nitric oxide synthase gene expression

L-buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH synthesis, decreased IL-1 beta-induced nitrite release in rat islets and purified rat beta cells, nitrite formation and iNOS gene promoter activity in insulinoma cells, and iNOS mRNA expression in rat islets. The thiol depletor diethyl maleate (DEM) and an inhibitor of glutathione reductase 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) reduced IL-1 beta-stimulated nitrite release in islets. We conclude that GSH regulates IL-1 beta-induced NO production in islets, purified beta cells and insulinoma cells by modulation of iNOS gene expression.     

Inhaled nitric oxide increases surfactant protein gene expression in the intact lamb

Inhaled nitric oxide (iNO) is used to treat a number of disease processes. Although in vitro data suggest that nitric oxide (NO) alters surfactant protein gene expression, the effects in vivo have not been studied. The objective of this study was to evaluate the effects of iNO on surfactant protein (SP)-A, -B, and -C gene expression in the intact lamb. Thirteen 4-wk-old lambs were mechanically ventilated with 21% oxygen and received iNO at 40 ppm (n = 7) or vehicle gas (n = 6) for 24 h. Peripheral lung biopsies were obtained at 0, 12, and 24 h and analyzed for surfactant mRNA, protein, and total DNA content. Inhaled NO increased SP-A and SP-B mRNA content by 80% from 0 to 12 h and by 78 and 71%, respectively, from 0 to 24 h. There was an increase in SP-A and SP-B protein content by 45% from 0 to 12 h, and a decrease by 70 and 65%, respectively, from 0 to 24 h. DNA content was unchanged. The mechanisms and physiological effects of these findings warrant further investigation.     

D1 protein degradation during photoinhibition of intact leaves a modification of the D1 protein precedes degradation

Illumination of intact pumpkin leaves with high light led to severe photoinhibition of photosystem II with no net degradation of the D1 protein. Instead, however, a modified form of D1 protein with slightly slower electrophoretic mobility was induced with corresponding loss in the original form of the D1 protein. When the leaves were illuminated in the presence of chloramphenicol the modified form was degraded, which led to a decrease in the total amount of the D1 protein. Subfractionation of the thylakoid membranes further supported the conclusion that the novel form of the D1 protein was not a precursor but a high-light modified form that was subsequently degraded.     

Arabidopsis nonsymbiotic hemoglobin AHb1 modulates nitric oxide bioactivity

Nitric oxide (NO) is a widespread signaling molecule, and numerous targets of its action exist in plants. Whereas the activity of NO in erythrocytes, microorganisms, and invertebrates has been shown to be regulated by several hemoglobins, the function of plant hemoglobins in NO detoxification has not yet been elucidated. Here, we show that Arabidopsis thaliana nonsymbiotic hemoglobin AHb1 scavenges NO through production of S-nitrosohemoglobin and reduces NO emission under hypoxic stress, indicating its role in NO detoxification. However, AHb1 does not affect NO-mediated hypersensitive cell death in response to avirulent Pseudomonas syringae, suggesting that it is not involved in the removal of NO bursts originated from acute responses when NO mediates crucial defense signaling functions.     

Increased ubiquitin-dependent degradation can replace the essential requirement for heat shock protein induction

Serine palmitoyltransferase, the first enzyme in ceramide biosynthesis, is required for resistance to heat shock. We show that increased heat shock sensitivity in the absence of serine palmitoyltransferase activity correlates with a lack of induction of the major heat shock proteins (Hsps) at high temperature. Normal heat shock resistance can be restored, without restoration of ceramide synthesis or induction of Hsps, by overexpression of ubiquitin. This function of ubiquitin requires the proteasome. These data imply that the essential function of Hsp induction is the removal of misfolded or aggregated proteins, not their refolding. This suggests that cells stressed by heat shock do not die because of the loss of protein activity due to their denaturation, but because of the inherent toxicity of the denatured and/or aggregated proteins.     

An F-box protein, FWD1, mediates ubiquitin-dependent proteolysis of beta-catenin

beta-catenin plays an essential role in the Wingless/Wnt signaling cascade and is a component of the cadherin cell adhesion complex. Deregulation of beta-catenin accumulation as a result of mutations in adenomatous polyposis coli (APC) tumor suppressor protein is believed to initiate colorectal neoplasia. beta-catenin levels are regulated by the ubiquitin-dependent proteolysis system and beta-catenin ubiquitination is preceded by phosphorylation of its N-terminal region by the glycogen synthase kinase-3beta (GSK-3beta)/Axin kinase complex. Here we show that FWD1 (the mouse homologue of Slimb/betaTrCP), an F-box/WD40-repeat protein, specifically formed a multi-molecular complex with beta-catenin, Axin, GSK-3beta and APC. Mutations at the signal-induced phosphorylation site of beta-catenin inhibited its association with FWD1. FWD1 facilitated ubiquitination and promoted degradation of beta-catenin, resulting in reduced cytoplasmic beta-catenin levels. In contrast, a dominant-negative mutant form of FWD1 inhibited the ubiquitination process and stabilized beta-catenin. These results suggest that the Skp1/Cullin/F-box protein FWD1 (SCFFWD1)-ubiquitin ligase complex is involved in beta-catenin ubiquitination and that FWD1 serves as an intracellular receptor for phosphorylated beta-catenin. FWD1 also links the phosphorylation machinery to the ubiquitin-proteasome pathway to ensure prompt and efficient proteolysis of beta-catenin in response to external signals. SCFFWD1 may be critical for tumor development and suppression through regulation of beta-catenin protein stability.     

Shp1 and Ubx2 are adaptors of Cdc48 involved in ubiquitin-dependent protein degradation

Known activities of the ubiquitin-selective AAA ATPase Cdc48 (p97) require one of the mutually exclusive cofactors Ufd1/Npl4 and Shp1 (p47). Whereas Ufd1/Npl4 recruits Cdc48 to ubiquitylated proteins destined for degradation by the 26S proteasome, the UBX domain protein p47 has so far been linked exclusively to nondegradative Cdc48 functions in membrane fusion processes. Here, we show that all seven UBX domain proteins of Saccharomyces cerevisiae bind to Cdc48, thus constituting an entire new family of Cdc48 cofactors. The two major yeast UBX domain proteins, Shp1 and Ubx2, possess a ubiquitin-binding UBA domain and interact with ubiquitylated proteins in vivo. Deltashp1 and Deltaubx2 strains display defects in the degradation of a ubiquitylated model substrate, are sensitive to various stress conditions and are genetically linked to the 26S proteasome. Our data suggest that Shp1 and Ubx2 are adaptors for Cdc48-dependent protein degradation through the ubiquitin/proteasome pathway.     

AtSERK1 expression precedes and coincides with early somatic embryogenesis in Arabidopsis thaliana.

The Arabidopsis thaliana primordia timing (pt) mutant was transformed with an AtSERK1::GUS construct. Liquid cultures of this line were used to study the relationship between somatic embryogenesis and the expression of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (AtSERK1) as a marker for cells competent to form embryos. In order to search for the expression of AtSERK1::GUS during early stages of somatic embryogenesis, histochemical as well as immunochemical approaches were used for the detection of beta-glucuronidase (GUS). Four sites of AtSERK1 expression were found in the embryogenic cultures: in embryogenic callus, where primary somatic embryos developed; in the basal parts of primary somatic embryos; in the outer layers of cotyledons of primary somatic embryos where secondary embryos were formed; and in provascular and vascular strands of developing somatic embryos. The in vitro expression of AtSERK1::GUS coincides with embryogenic development up to the heart-shaped stage. Prior to the expression in embryos, AtSERK1 was expressed in single cells and small cell clusters, indicating that AtSERK1 indeed marks embryogenic competence. Its expression in (pro)vascular strands, suggests that embryogenic cells in tissue culture retain at least in part their original identity.