Distinct UV-B and UV-A/blue light signal transduction pathways induce chalcone synthase gene expression in Arabidopsis cells

UV and blue light control the expression of flavonoid biosynthesis genes in a range of higher plants. To investigate the signal transduction processes involved in the induction of chalcone synthase (CHS) gene expression by UV-B and UV-A/blue light, we examined the effects of specific agonists and inhibitors of known signaling components in mammalian systems in a photomixotrophic Arabidopsis cell suspension culture. CHS expression is induced specifically by these wavelengths in the cell culture, in a manner similar to that in mature Arabidopsis leaf tissue. Both the UV-B and UV-A/blue phototransduction processes involve calcium, although the elevation of cytosolic calcium is insufficient on its own to stimulate CHS expression. The UV-A/blue light induction of CHS expression does not appear to involve calmodulin, whereas the UV-B response does; this difference indicates that the signal transduction pathways are, at least in part, distinct. We provide evidence that both pathways involve reversible protein phosphorylation and require protein synthesis. The UV-B and UV-A/blue light signaling pathways are therefore different from the phytochrome signal transduction pathway regulating CHS expression in other species.     

Involvement of plasma membrane redox activity and calcium homeostasis in the UV-B and UV-A/blue light induction of gene expression in Arabidopsis

UV and blue light are important regulators of plant gene expression and development. We investigated the signal transduction processes involved in the induction of chalcone synthase (CHS) and phenylalanine ammonia-lyase (PAL) gene expression by UV-B and UV-A/blue light in an Arabidopsis cell suspension culture. Experiments with electron transport inhibitors indicated that plasma membrane redox activity is involved in both signal transduction pathways. Calcium ionophore treatment stimulated expression of the TOUCH3 gene, and this induction was strongly antagonized by UV-A/blue and UV-B light, suggesting that both light qualities may promote calcium efflux from the cytosol. Consistent with this hypothesis, experiments with specific inhibitors indicated that UV-B and UV-A/blue light regulate calcium levels in a cytosolic pool in part via the action of specific Ca2+-ATPases. On the basis of these and previous findings, we propose that plasma membrane redox activity, initiated by photoreception, is coupled to the regulation of calcium release from an intracellular store, generating a calcium signal that is required to induce CHS expression.     

Reactive oxygen species and antioxidants in signal transduction and gene expression

Reactive oxygen species (ROS) are produced by cellular metabolic reactions, and have been implicated in the pathogenesis of several diseases, including atherosclerosis, cancer, and Alzheimer's disease. Interestingly, clinical and epidemiologic studies have, in some cases, indicated that antioxidant nutrients may be effective in disease prevention. However, the efficacy of specific antioxidants in disease prevention is often both controversial and inconclusive. In an effort to elucidate the role of ROS and antioxidants in disease development and prevention, the chemistries of ROS and antioxidants have been examined extensively. Recently, molecular and cellular approaches have demonstrated that ROS and antioxidants can directly affect the cellular signaling apparatus and, consequently, the control of gene expression. This new research provides the link between ROS and antioxidant chemistries and the mechanisms of disease processes and prevention. This review illustrates how ROS function as potential intracellular and extracellular signaling molecules and how antioxidants can affect this process.     

The use of dominant-negative mutations to elucidate signal transduction pathways in lymphocytes

A simple procedure for the determination of amphetamine in urine with minimal sample preparation is described. This method involves direct addition of human urine to an acetone-dansyl chloride solution for simultaneous deproteinization and fluorescence derivatization. The derivatized amphetamine is then measured by HPLC with fluorescence detection. It eliminates the extraction procedures often required by other HPLC or GC methods. The effects of pH, temperature and reaction time on the derivatization reaction were investigated. The stability of amphetamine-dansyl chloride in different storage conditions was examined. The detection limit and linearity associated with this assay are discussed.     

Two distinct, calcium-mediated, signal transduction pathways can trigger deflagellation in Chlamydomonas reinhardtii

The molecular machinery of deflagellation can be activated in detergent permeabilized Chlamydomonas reinhardtii by the addition of Ca2+ (Sanders, M. A., and J. L. Salisbury, 1989. J. Cell Biol. 108:1751-1760). This suggests that stimuli which induce deflagellation in living cells cause an increase in the intracellular concentration of Ca2+, but this has never been demonstrated. In this paper we report that the wasp venom peptide, mastoparan, and the permeant organic acid, benzoate, activate two different signalling pathways to trigger deflagellation. We have characterized each pathway with respect to: (a) the requirement for extracellular Ca2+; (b) sensitivity to Ca2+ channel blockers; and (c) 45Ca influx. We also report that a new mutant strain of C. reinhardtii, adf-1, is specifically defective in the acid-activated signalling pathway. Both signalling pathways appear normal in another mutant, fa-1, that is defective in the machinery of deflagellation (Lewin, R. and C. Burrascano. 1983. Experientia. 39:1397-1398; Sanders, M. A., and J. L. Salisbury. 1989. J. Cell Biol. 108:1751-1760). We conclude that mastoparan induces the release of an intracellular pool of Ca2+ whereas acid induces an influx of extracellular Ca2+ to activate the machinery of deflagellation.     

Biotin synthase from Arabidopsis thaliana. cDNA isolation and characterization of gene expression

The full-length BIO2 cDNA from Arabidopsis thaliana was isolated using an expressed sequence tag that was homologous to the Escherichia coli biotin synthase gene (BioB). Comparisons of the deduced amino acid sequence from BIO2 with bacterial and yeast biotin synthase homologs revealed a high degree of sequence similarity. The amino terminus of the predicted BIO2 protein contains a stretch of hydrophobic residues similar in composition to transit peptide sequences. BIO2 is a single-copy nuclear gene in Arabidopsis that is expressed at high levels in the tissues of immature plants. Expression of BIO2 was higher in the light relative to dark and was induced 5-fold during biotin-limited conditions. These results demonstrate that expression of at least one gene in this pathway is regulated in response to developmental, environmental, and bio-chemical stimuli.     

STAR, a gene family involved in signal transduction and activation of RNA

A new subfamily of KH-domain-containing RNA-binding proteins is encoded by genes that are conserved from yeast to humans. Mutations with interesting developmental phenotypes have been identified in Caenorhabditis elegans, Drosophila and mouse. It is hypothesized that these bifunctional proteins provide a rich source of interesting molecular information about development and define a new cellular pathway that links signal transduction directly to RNA metabolism.     

Metabolic oxidative stress activates signal transduction and gene expression during glucose deprivation in human tumor cells

The mechanism of glucose deprivation-induced activation of Lyn kinase (Lyn), c-Jun N-terminal kinase 1 (JNK1) and increased expression of basic fibroblast growth factor (bFGF) and c-Myc was investigated in MCF-7/ADR adriamycin-resistant human breast carcinoma cells. Glucose deprivation significantly increased steady state levels of oxidized glutathione content (GSSG) and intracellular prooxidants (presumably hydroperoxides) as well as caused the activation of Lyn, JNK1, and the accumulation of bFGF and c-Myc mRNA. The suppression of GSSG accumulation and prooxidant production by treatment with the thiol antioxidant, N-acetylcysteine, also suppressed all the increases in kinase activation and gene expression observed during glucose deprivation. In addition, glucose deprivation was shown to induce oxidative stress in IMR90 SV40 transformed human fibroblasts, indicating that this phenomena is not limited to the MCF-7/ADR cell line. These and previous observations from our laboratory show that glucose deprivation-induced oxidative stress in MCF-7/ADR cells activates signal transduction involving Lyn, JNK1, and mitogen activated protein kinases (ERK1/ERK2) which results in increased bFGF and c-Myc mRNA accumulation. These results provide support for the hypothesis that alterations in intracellular oxidation/reduction reactions link changes in glycolytic metabolism to signal transduction and gene expression in these human tumor cells.     

Tubulin, Gq, and phosphatidylinositol 4,5-bisphosphate interact to regulate phospholipase Cbeta1 signaling

The cytoskeletal protein, tubulin, has been shown to regulate adenylyl cyclase activity through its interaction with the specific G protein alpha subunits, Galphas or Galphai1. Tubulin activates these G proteins by transferring GTP and stabilizing the active nucleotide-bound Galpha conformation. To study the possibility of tubulin involvement in Galphaq-mediated phospholipase Cbeta1 (PLCbeta1) signaling, the m1 muscarinic receptor, Galphaq, and PLCbeta1 were expressed in Sf9 cells. A unique ability of tubulin to regulate PLCbeta1 was observed. Low concentrations of tubulin, with guanine nucleotide bound, activated PLCbeta1, whereas higher concentrations inhibited the enzyme. Interaction of tubulin with both Galphaq and PLCbeta1, accompanied by guanine nucleotide transfer from tubulin to Galphaq, is suggested as a mechanism for the enzyme activation. The PLCbeta1 substrate, phosphatidylinositol 4,5-bisphosphate, bound to tubulin and prevented microtubule assembly. This observation suggested a mechanism for the inhibition of PLCbeta1 by tubulin, since high tubulin concentrations might prevent the access of PLCbeta1 to its substrate. Activation of m1 muscarinic receptors by carbachol relaxed this inhibition, probably by increasing the affinity of Galphaq for tubulin. Involvement of tubulin in the articulation between PLCbeta1 signaling and microtubule assembly might prove important for the intracellular governing of a broad range of cellular events.     

Anion channels and the stimulation of anthocyanin accumulation by blue light in Arabidopsis seedlings

The effects of endothelin 1 (ET-1) on hemodynamics and acute liver damage were studied using perfused livers of rats treated with D-galactosamine. In control liver perfused in situ with constant pressure, infusion of ET-1 into the portal vein at a concentration of 0.1 nmol/L decreased the flow rate without a significant leakage of lactate dehydrogenase (LDH) or aspartate transaminase (AST) into the effluent. In contrast, in similarly perfused liver 24 hours after treatment with D-galactosamine (800 mg/kg intraperitoneally), ET-1 caused rapid and remarkable increases in the leakage of LDH and AST from the liver accompanied by the reduction of perfusion flow to the extent similar to that observed in control livers. In addition, ET-1 decreased oxygen uptake and bile secretion in galactosamine-treated livers. The potentiating effects of ET-1 on enzyme leakage were also observed under constant flow conditions. Moreover, infusion of the thromboxane A2 analogue at a concentration of 10 nmol/L decreased the flow rate markedly, yet the rapid increases in enzyme leakage were not observed. Infusion of ET-3 induced the responses of flow reduction and the potentiation of rapid enzyme leakage similar to those obtained with ET-1. Neither the endothelin A-receptor antagonist BQ485 nor the endothelin B-receptor antagonist BQ788 could inhibit the acute liver damage caused by ET-1; instead they exaggerated its effects. The combination of both antagonists together, however, almost completely suppressed the flow reduction and the potentiation of enzyme leakage caused by ET-1. These results indicate that ET-1 is capable of aggravating acute liver damage not merely through reduction of the flow rate but through direct action on liver cells. They also suggest that both the endothelin A and endothelin B receptors are involved in this action of ET-1.     

Wounding changes the spatial expression pattern of the arabidopsis plastid omega-3 fatty acid desaturase gene (FAD7) through different signal transduction pathways

The Arabidopsis FAD7 gene encodes a plastid omega-3 fatty acid desaturase that catalyzes the desaturation of dienoic fatty acids in membrane lipids. The mRNA levels of the Arabidopsis FAD7 gene in rosette leaves rose rapidly after local wounding treatments. Wounding also induced the expression of the FAD7 gene in roots. To study wound-responsive expression of the FAD7 gene in further detail, we analyzed transgenic tobacco plants carrying the -825 Arabidopsis FAD7 promoter-beta-glucuronidase fusion gene. In unwounded transformants, FAD7 promoter activity was restricted to the tissues whose cells contained chloroplasts. Activation of the FAD7 promoter by local wounding treatments was more substantial in stems (29-fold) and roots (10-fold) of transgenic plants than it was in leaves (approximately two-fold). Significant induction by wounding was observed in the overall tissues of stems and included trichomes, the epidermis, cortex, vascular system, and the pith of the parenchyma. Strong promoter activity was found preferentially in the vascular tissues of wounded roots. These results indicate that wounding changes the spatial expression pattern of the FAD7 gene. Inhibitors of the octadecanoid pathway, salicylic acid and n-propyl gallate, strongly suppressed the wound activation of the FAD7 promoter in roots but not in leaves or stems. In unwounded plants, exogenously applied methyl jasmonate activated the FAD7 promoter in roots, whereas it repressed FAD7 promoter activity in leaves. Taken together, wound-responsive expression of the FAD7 gene in roots is thought to be mediated via the octadecanoid pathway, whereas in leaves, jasmonate-independent wound signals may induce the activation of the FAD7 gene. These observations indicate that wound-responsive expression of the FAD7 gene in aerial and subterranean parts of plants is brought about by way of different signal transduction pathways.     

Repression of c-fos and c-jun gene expression is not part of AT2 receptor coupled signal transduction

Recent world-wide publications were reviewed in order to determine the clinical characteristics and therapeutic relevance of the chlamydial respiratory tract infections in humans. It was emphasized that Chlamydia pneumoniae could initiate asthma and may be associated with acute asthma exacerbation. Laboratory procedures for identifying chlamydia and difficulties concerned with the diagnostics of this intracellular pathogen were also presented. In patients with evidence of chlamydial infection the casual treatment (macrolides, tetracyclines, fluoroquinolones) may induce major improvement or complete resolution of asthma.