Protoplasts derived from cell suspensions of alfalfa (Medicago sativa L.) responded to treatment with fungal elicitor (FE) by an increase in endogenous chalcone synthase (CHS) activity but were unresponsive to reduced glutathione (GSH). Preexposure of protoplasts to polyethylene glycol and electroporation resulted in strong responsiveness to GSH but little change in responsiveness to FE. Protoplasts from suspension cultures which had been subcultured more than 12 times lost responsiveness to GSH, but not FE, as assessed by measuring expression of a chimeric gene containing a bean CHS promoter linked to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene. In protoplasts in which putative cis-acting CHS promoter sequences had been coelectroporated in trans with the intact CHS promoter-CAT construct, the extent of CAT expression depended upon the elicitor used (FE or GSH), the age (number of times subcultured) of the cells from which the protoplasts were isolated, and the nature of the coelectroporated CHS promoter sequence. For example, a region of the CHS promoter from -326 to -141 behaved as a trans-activator when coelectroporated with the CAT construct into unelicited protoplasts isolated from newly initiated cell suspensions, but the same region acted as a trans-silencer in the same protoplasts in the presence of FE. This silencer activity was much reduced in GSH-treated protoplasts. The results suggest that there are differences in the signal transduction pathways for elicitation of CHS transcription by FE and GSH, which involve previously identified cis-elements in the CHS promoter. A wide range of structurally diverse biotic and abiotic agents (elicitors) have been shown to induce the expression of defense metabolism in plants (3, 4). This raises the obvious question of whether they act by a common mechanism. Of particular interest is the observation that reduced GSH acts as a strong elicitor of defense gene activation in intact suspension cells and protoplasts of bean and soybean (7, 13), selectively inducing gene expression in a manner similar to that observed in response to fungal elicitor preparations from the cell walls of the bean pathogen Colletotrichum lindemuthianum. A quantitatively major set of transcripts induced by GSH in cultured bean cells (13) and pea epicotyl tissue (14) encodes CHS2, a key regulatory enzyme in the biosynthesis of isoflavonoid phytoalexins in legume cells. CHS enzyme activity is also strongly induced by GSH in suspension cultured cells of the cactus Cephalocereus senilis (P. Paray, personal communication). CHS is encoded by a family of at least six genes in
The interaction of trans ‐cinnamic acid with the cytochrome P ‐450 of microsomes derived from washed potato slices has been studied. The washing process increased the specific content of microsomal electron transport components and hence provided a useful material in which to study the interaction. Evidence is presented that the trans ‐cinnamic acid interacts with the cytochrome P ‐450, and that this interaction is analogous to ‘type I’ interactions of other cytochrome P ‐450 systems. This evidence includes the formation of a ‘type I’ substrate binding spectrum, an increased rate of reduction of cytochrome P ‐450 by NADPH in the presence of trans ‐cinnamic acid, an increased oxygen uptake and NADPH oxidation when trans ‐cinnamic acid is added to the microsomes in the presence of NADPH, and a close correlation between biophysical parameters of electron transport in the cytochrome P ‐450 system and enzymological parameters of the trans ‐cinnamic acid 4‐hydroxylation reaction. The investigation has been extended to cytochrome P ‐450 systems of other tissues and it has been found that the trans ‐cinnamic acid 4‐hydroxylation reaction cannot account for the presence of most of the cytochrome P ‐450 in several tissues. This suggests that other functions of higher plant cytochrome P ‐450 chains exist, and that the substrate specificity of the hemoprotein may vary in different plant tissues.
Crop loss due to soil salinization is an increasing threat to agriculture worldwide. This review provides an overview of cellular and physiological mechanisms in plant responses to salt. We place cellular responses in a time- and tissue-dependent context ...Read More
A 46-year-old, right-handed, African American man presented to the emergency department after 1 week of persistent hiccups and 3 days of nausea and vomiting. His family member also noted that he had been experiencing right-hand tremor and gait imbalance for the last 3 months. Over the 6 months prior to presentation, the patient's coworkers had noticed that he was completing his work more slowly, that he was forgetting how to perform simple tasks, and that several times he had come to work at the wrong hours. The patient had a history of HIV diagnosed 10 years earlier and reported good adherence to an antiretroviral regimen consisting of tenofovir, emtricitabine, and atazanavir boosted by ritonavir, without any changes for several years. His viral load was undetectable when tested 8 months prior to presentation.
The bean phenylalanine ammonia-lyase gene 2 (PAL2) is expressed in the early stages of vascular development at the inception of xylem differentiation, associated with the synthesis of lignin precursors.This is part of a complex program of developmental expression regulating the synthesis of functionally diverse phenylpropanoid natural products.Analysis of the expression of PAL2 promoter-P-glucuronidase gene fusions in transgenic tobacco plants showed that functionally redundant cis elements located between nucleotides -289 and -74 relative to the transcription start site were essential for xylem expression, but were not involved in expression in leaf primordia and stem nodes or in establishing tissue specificity in petals.The -135 to -119 region implicated in xylem expression contains a negative element that suppresses the activity of a cryptic cis element for phloem expression located between -480 and -289.The functional properties of each vascular element are conserved in stem, petiole, and root, even though the xylem and phloem are organized in different patterns in these organs.We conclude that the PAL2 promoter has a modular organization and that tissue-specific expression in the vascular system involves a negative combinatorial interaction, modulation of which may provide a flexible mechanism for modification of tissue specificity. INTRODUMIONPhenylalanine ammonia-lyase (PAL; EC 4.3.1.5)catalyzes the first reaction in the synthesis from phenylalanine of a wide range of natural products based on the phenylpropane skeleton, including cinnamyl alcohols, which are the substrates for the peroxidase-mediated oxidative reactions that generate the lignin polymer (Lamb et al., 1989).Dissection of stems reveals high levels of PAL enzyme activity specifically in vascular tissues (Rubery and Northcote, 1968), and the induction of xylem differentiation in callus culture by manipulation of auxin levels is accompanied by a marked increase in PAL activity (Haddon and Northcote, 1975).These data suggest that PAL is a key regulatory step in lignin synthesis during xylogenesis, and this has recently been confirmed by gene transfer experiments in which a reduction of PAL activity in transgenic tobacco plants, engineered by introduction of heterologous PAL
l ‐Phenylalanine ammonia‐lyase (EC 4.3.1.9, which catalyses the first reaction in the biosynthesis of plant phenylpropanoid products, undergoes a transient increase in activity in excised sections of pea ( Pisum sativum ) epicotyl tissue. Exogenous supplies of pathway intermediates inhibit the initial development of phenylalanine ammonia‐lyase activity and, if added at the time of high enzyme levels, cause a rapid decay in enzyme activity. The inhibitory effect of exogenous cinnamic acid, the immediate product of the enzyme, is expressed very rapidly (lag < 1 h) with the initial rate of decay of enzyme activity and the final steady‐state level of enzyme activity being dependent on the concentration of cinnamic acid. The response has been studied by density labelling in vivo with 2 H from 2 H 2 O with analysis of the equilibrium distribution of enzyme activity in high‐resolution KBr density gradients. Cinnamic acid, at a concentration of 1 mM completely inhibits further incorporation of label and also markedly stimulates the removal of pre‐existing, unlabelled enzyme. Inhibition of de novo production and stimulation of removal occur at concentrations of cinnarnic acid as low as 10 μM. The initial increase in enzyme activity reflects an increase in the rate of de novo enzyme production against a low background rate of enz:yme removal. The subsequent decay in enzyme activity is a result of a decline in production of the enzyme concomitant with a marked increase in the rate of removal of enzyme activity. In the presence of α‐ aminooxy‐β‐phenylpropionic acid, which specifically inhibits phenylalanine ammonia‐lyase and hence endogenous generation of cinnamic cid, high rates of enzyme production and low rates of enzyme removal are maintained leading to high levels of zyme activity in the later stages of enzyme induction. These data indicate the operation of dual feedback modulation of phenylalanine ammonia‐lyase in vivo following endogenous production of cinnamic acid. Such dual control by a product over both production and removal of an enzyme may be of general importance in slowly growing plant cells for the rapid removal of biosynthetic enzymes no longer required by the cell.
