Suppression of CaCYP1, a novel cytochrome P450 gene, compromises the basal pathogen defense response of pepper plants

A putative cytochrome P450 gene from chili pepper, Capsicum annuum L. Bukang cytochrome P450 (CaCYP1), was identified using cDNA microarray analysis of gene expression following induction of the leaf hypersensitive response by inoculation of pepper plants with the non-host pathogen Xanthomonas axonopodis pv. glycines 8ra. The full-length cDNA of CaCYP1 encoded a protein of 514 amino acid residues, which contained a putative hydrophobic membrane anchoring domain in the N-terminal region, and a heme-binding motif in the C-terminal region. Analysis of the deduced amino acid sequence of CaCYP1 revealed that it has high homology to Arabidopsis CYP89A5, the function of which is unknown. Expression of CaCYP1 was preferentially increased in pepper plants in response to non-host pathogen inoculation and also during the host resistance response. CaCYP1 expression also increased following treatment with salicylic acid and abscisic acid, while treatment with ethylene had a mild effect. Using a virus-induced gene silencing-based reverse genetics approach, we demonstrated that suppression of CaCYP1 results in enhanced susceptibility to bacterial pathogens. Interestingly, gene silencing of CaCYP1 in pepper plants resulted in the reduced expression of the defense-related genes CaLTP1, CaSIG4, and Cadhn. Our results indicated that CaCYP1, a novel cytochrome P450 in pepper plants, may play a role in plant defense response pathways that involve salicylic acid and abscisic acid signaling pathways.     

The pepper mannose-binding lectin gene CaMBL1 is required to regulate cell death and defense responses to microbial pathogens

Plant mannose-binding lectins (MBLs) are crucial for plant defense signaling during pathogen attack by recognizing specific carbohydrates on pathogen surfaces. In this study, we isolated and functionally characterized a novel pepper (Capsicum annuum) MBL gene, CaMBL1, from pepper leaves infected with Xanthomonas campestris pv vesicatoria (Xcv). The CaMBL1 gene contains a predicted Galanthus nivalis agglutinin-related lectin domain responsible for the recognition of high-mannose N-glycans but lacks a middle S-locus glycoprotein domain and a carboxyl-terminal PAN-Apple domain. The CaMBL1 protein exhibits binding specificity for mannose and is mainly localized to the plasma membrane. Immunoblotting using a CaMBL1-specific antibody revealed that CaMBL1 is strongly expressed and accumulates in pepper leaves during avirulent Xcv infection. The transient expression of CaMBL1 induces the accumulation of salicylic acid (SA), the activation of defense-related genes, and the cell death phenotype in pepper. The G. nivalis agglutinin-related lectin domain of CaMBL1 is responsible for cell death induction. CaMBL1-silenced pepper plants are more susceptible to virulent or avirulent Xcv infection compared with unsilenced control plants, a phenotype that is accompanied by lowered reactive oxygen species accumulation, reduced expression of downstream SA target genes, and a concomitant decrease in SA accumulation. In contrast, CaMBL1 overexpression in Arabidopsis (Arabidopsis thaliana) confers enhanced resistance to Pseudomonas syringae pv tomato and Alternaria brassicicola infection. Together, these data suggest that CaMBL1 plays a key role in the regulation of plant cell death and defense responses through the induction of downstream defense-related genes and SA accumulation after the recognition of microbial pathogens.     

Phytoalexins in defense against pathogens

Plants use an intricate defense system against pests and pathogens, including the production of low molecular mass secondary metabolites with antimicrobial activity, which are synthesized de novo after stress and are collectively known as phytoalexins. In this review, we focus on the biosynthesis and regulation of camalexin, and its role in plant defense. In addition, we detail some of the phytoalexins produced by a range of crop plants from Brassicaceae, Fabaceae, Solanaceae, Vitaceae and Poaceae. This includes the very recently identified kauralexins and zealexins produced by maize, and the biosynthesis and regulation of phytoalexins produced by rice. Molecular approaches are helping to unravel some of the mechanisms and reveal the complexity of these bioactive compounds, including phytoalexin action and metabolism.     

Regulation of cytochrome P450 gene expression in the olfactory mucosa

The mammalian olfactory mucosa (OM) is unique among extrahepatic tissues in having high levels, and tissue-selective forms, of cytochrome P450 (CYP) enzymes. These enzymes may have important toxicological implications, as well as biological functions, in this chemosensory organ. In addition to a tissue-selective, abundant expression of CYP1A2, CYP2A, and CYP2G1, some of the OM CYPs are also known to have an early developmental expression, a resistance to xenobiotic inducers, and a lack of responsiveness to circadian rhythm. Efforts to fully characterize the regulation of CYP expression in the OM, and to identify the underlying mechanisms, are important for our understanding of the physiological functions and toxicological significance of these biotransformation enzymes, and may also shed unique light on the general mechanisms of CYP regulation. The aim of this mini-review is to provide a summary of current knowledge of the various modes of regulation of CYPs expressed in the OM, an update on our mechanistic studies on tissue-selective CYP expression, and a review of the literature on xenobiotic inducibility of OM CYPs. Our goal is to stimulate further studies in this exciting research area, which is of considerable importance, in view of the constant exposure of the human nasal tissues to inhaled, as well as systemically derived, chemicals, the prevalence of olfactory system damage in individuals with neurodegenerative diseases, and the current uncertainty in risk assessments for potential olfactory toxicants.     

The role of cytochrome P450 monooxygenases in microbial fatty acid metabolism

Cytochrome P450 monooxygenases (P450s) are a diverse collection of enzymes acting on various endogenous and xenobiotic molecules. Most of them catalyse hydroxylation reactions and one group of possible substrates are fatty acids and their related structures. In this minireview, the significance of P450s in microbial fatty acid conversion is described. Bacteria and yeasts possess various P450 systems involved in alkane and fatty acid degradation, and often several enzymes with different activities and specificities are retrieved in one organism. Furthermore, P450s take part in the formation of fatty acid-based secondary metabolites. Finally, there are a substantial number of microbial P450s displaying activity towards fatty acids, but to which no biological role could be assigned despite the often quite intense research.     

The role of sugar signaling in plant defense responses against fungal pathogens

In most fungal pathogen–plant systems, a high level of sugars in plant tissues enhances plant resistance. Several hypotheses have been proposed to explain the mechanisms of “high-sugar resistance”. Sugars constitute the primary substrate providing energy and structural material for defense responses in plants, while they may also act as signal molecules interacting with the hormonal signaling network regulating the plant immune system. Sugars enhance oxidative burst at early stages of infection, increasing lignification of cell walls, stimulate the synthesis of flavonoids and induce certain PR proteins. Some sugars act as priming agents inducing higher plant resistance to pathogens.     

Expression of a cytochrome P450 gene family in maize

Maize seedlings, like seedlings of many other plants, are rich in cytochrome P450 (P450) enzyme activity. Four P450 genes (CYPzm1–4), isolated from a seedling-specific cDNA library, are characterised by a transient and seedling-specific expression pattern. The maximum steady state mRNA levels are reached at 3 days in root and at 7 days in shoot tissue, respectively. All four genes belong to one gene family and are closely related to the CYP71 family of plant P450 genes, which includes the enzymes of the ripening avocado fruit (CYP71A1) and eggplant hypocotyls (CYP71A2, A3, A4). The expression of these related P450 genes in monocot and dicot plants indicates that these enzymes play a significant role in plants; however, the in vivo enzyme functions are unknown. The divergence of the four members of the maize gene family is sufficiently high to account for different substrate and/or reaction specificity. Although the general expression pattern of the four genes is identical, the maximum steady-state mRNA levels vary in different maize lines. In situ hybridisation reveals the highest mRNA levels in the coleoptile, the first developed leaflets, the ground tissue of the nodular complex, and in the cortex and pith of the region of cell division in the root. The mapping of the maize CYPzm genes shows that, as in animals, P450 genes of the same family can be clustered. The presence of the CYPzm gene cluster in maize argues for generation of distinct plant P450 gene families by gene duplication.     

Role of the LolP cytochrome P450 monooxygenase in loline alkaloid biosynthesis

The insecticidal loline alkaloids, produced by Neotyphodium uncinatum and related endophytes, are exo-1-aminopyrrolizidines with an ether bridge between C-2 and C-7. Loline alkaloids vary in methyl, acetyl, and formyl substituents on the 1-amine, which affect their biological activity. Enzymes for key loline biosynthesis steps are probably encoded by genes in the LOL cluster, which is duplicated in N. uncinatum, except for a large deletion in lolP2. The role of lolP1 was investigated by its replacement with a hygromycin B phosphotransferase gene. Compared to wild type N. uncinatum and an ectopic transformant, DeltalolP1 cultures had greatly elevated levels of N-methylloline (NML) and lacked N-formylloline (NFL). Complementation of DeltalolP1 with lolP1 under control of the Emericella nidulans trpC promoter restored NFL production. These results and the inferred sequence of LolP1 indicate that it is a cytochrome P450, catalyzing oxygenation of an N-methyl group in NML to the N-formyl group in NFL.     

Role of c-Myc in nitric oxide-mediated suppression of cytochrome P450 3A4

Cytochrome P450 (CYP) 3A4, which is abundant in human liver and small intestine and participates in the metabolism of various drugs and xenochemicals, is known to be induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in the colon carcinoma cell line Caco-2 cells. Nitric oxide (NO) is able to inhibit CYP3A4 expression and catalytic activity. In this study, we investigated the mechanism of suppression by NO of 1,25(OH)2D3-induced CYP3A4 expression in Caco-2 cells. Caco-2 cells were exposed for 36 h to 400 nM 1,25(OH)2D3, and the induction of CYP3A4 mRNA expression was detected by real-time PCR. Because c-Myc regulates the expression of several genes, we examined its effect on the CYP3A4 expression induced by 1,25(OH)2D3. The expression of c-myc mRNA was increased in the early stage but decreased 36 h after the treatment of Caco-2 cells with 1,25(OH)2D3. The NO donor NOR-4 suppressed CYP3A4 expression induced by 1,25(OH)2D3 in Caco-2 cells in contrast, it significantly induced c-myc gene expression. Treatment of Caco-2 cells with the c-myc antisense oligonucleotide reversed the inhibitory effect of NOR-4 on CYP3A4 expression induced by 1,25(OH)2D3. These results suggest that the suppression of 1,25(OH)2D3-induced CYP3A4 expression by NO is due to c-myc expression.     

Role of cytochrome b5 in catalysis by cytochrome P450 2B4

Cytochrome b5 has been shown to stimulate, inhibit or have no effect on catalysis by P450 cytochromes. Its action is known to depend on the isozyme of cytochrome P450, the substrate, and experimental conditions. Cytochrome P450 2B4 (CYP 2B4) has been used in our laboratory as a model isozyme to study the role of cytochrome b5 in cytochrome P450 catalysis using two substrates, methoxyflurane and benzphetamine. One substrate is the volatile anesthetic, methoxyflurane, whose metabolism is consistently markedly stimulated by cytochrome b5. The other is benzphetamine, whose metabolism is minimally modified by cytochrome b5. Determination of the stoichiometry of the metabolism of both substrates showed that the amount of product formed is the net result of the simultaneous stimulatory and inhibitory actions of cytochrome b5 on catalysis. Site-directed mutagenesis studies revealed that both cytochrome b5 and cytochrome P450 reductase interact with cytochrome P450 on its proximal surface on overlapping but non-identical binding sites. Comparison of the rate of reduction of oxyferrous CYP 2B4 and the rate of substrate oxidation by cyt b5 and reductase with stopped-flow spectrophotometric and rapid chemical quench experiments has demonstrated that although cytochrome b5 and reductase reduce oxyferrous CYP 2B4 at the same rate, substrate oxidation proceeds more slowly in the presence of the reductase.     

Microsomal cytochrome P450 2C5: comparison to microbial P450s and unique features

Although microsomal P450s represent the majority of P450s, only microbial P450s have been amenable to crystal structure solution. We have recently solved the first crystal structure of a microsomal P450, 2C5, a progesterone hydroxylase from rabbit. We discuss the features of the protein in common with existing structures of microbial P450s and limitations of homology modeling mammalian P450s based on the microbial structures. Unique features involving membrane, substrate and cytochrome P450 reductase interactions are also discussed.     

A world of cytochrome P450s

The world we live in is a biosphere influenced by all organisms who inhabit it. It is also an ecology of genes, with some having rather startling effects. The premise put forth in this issue is cytochrome P450 is a significant player in the world around us. Life and the Earth itself would be visibly different and diminished without cytochrome P450s. The contributions to this issue range from evolution on the billion year scale to the colour of roses, from Darwin to Rachel Carson; all as seen through the lens of cytochrome P450.     

Role of cytochrome P450 in the control of the production of erythropoietin

Effects of agents affecting cytochrome P450 were studied on the production of erythropoietin (Epo) in cultures of the human hepatoma cell line HepG2. Epo was measured by radioimmunoassay of the culture media after 24 h of incubation. The addition of phenobarbital or 3-methylcholanthrene, which induce cytochrome P450, significantly enhanced the formation of Epo. Likewise, the thyroid hormones T3 and T4 stimulated the rate of the production of Epo. On the other hand, the formation of Epo was lowered following the addition of diethyldithiocarbamate or cysteamine chloride, which inhibit cytochrome P450. These findings support the idea that O2 sensitive hemoproteins of the microsomal mixed-functional oxidases play a role in the control of the synthesis of Epo.     

Role of cytochrome P450 in phospholipase A2- and arachidonic acid-mediated cytotoxicity

Phospholipases A2 (PLA2) comprise a set of extracellular and intracellular enzymes that catalyze the hydrolysis of the sn-2 fatty acyl bond of phospholipids to yield fatty acids and lysophospholipids. The PLA2 reaction is the primary pathway through which arachidonic acid (AA) is released from phospholipids. PLA2s have an important role in cellular death that occurs via necrosis or apoptosis. Several reports support the hypothesis that unesterified arachidonic acid in cells is a signal for the induction of apoptosis. However, most of the biological effects of arachidonic acid are attributable to its metabolism by mainly three different groups of enzymes: cytochromes P450, cyclooxygenases, and lipoxygenases. In this review we will focus on the role of cytochrome P450 in AA metabolism and toxicity. The major pathways of arachidonic acid metabolism catalyzed by cytochrome P450 generate metabolites that are subdivided into two groups: the epoxyeicosatrienoic acids, formed by CYP epoxygenases, and the arachidonic acid derivatives that are hydroxylated at or near the omega-terminus by CYP omega-oxidases. In addition, autoxidation of AA by cytochrome P450-derived reactive oxygen species produces lipid hydroperoxides as primary oxidation products. In some cellular models of toxicity, cytochrome P450 activity exacerbates PLA2- and AA-dependent injury, mainly through the production of oxygen radicals that promote lipid peroxidation or production of metabolites that alter Ca2+ homeostasis. In contrast, in other situations, cytochrome P450 metabolism of AA is protective, mainly by lowering levels of unesterified AA and by production of metabolites that activate antiapoptotic pathways. Several lines of evidence point to the combined action of phospholipase A2 and cytochrome P450 as central in the mechanism of cellular injury in several human diseases, such as alcoholic liver disease and myocardial reperfusion injury. Inhibition of specific PLA2 and cytochrome P450 isoforms may represent novel therapeutic strategies against these diseases.     

Characterization of the alkane-inducible cytochrome P450 (P450alk) gene from the yeast Candida tropicalis: identification of a new P450 gene family

The P450alk gene, which is inducible by the assimilation of alkane in Candida tropicalis, was sequenced and characterized. Structural features described in promoter and terminator regions of Saccharomyces yeast genes are present in the P450alk gene and some particular structures are discussed for their possible role in the inducibility of this gene. Expression of the P450alk gene was achieved in Saccharomyces cerevisiae using the yeast alcohol dehydrogenase expression system after removal of the P450alk gene flanking regions. The resultant expressed protein had a molecular mass slightly greater than that of P450alk from C. tropicalis. This alteration did not prevent the function and the localization of P450alk expressed in S. cerevisiae, as this organism showed an acquired microsome-bound activity for the terminal hydroxylation of lauric acid. The deduced P450alk amino acid sequence was compared with members of the nine known P450 gene families. These comparisons indicated that P450alk had a low relationship with these members and was therefore the first member (A1) of a new P450 gene family (LII).     

Emerging roles in plant defense for cis-jasmone-induced cytochrome P450 CYP81D11

Cis-jasmone is a volatile organic compound emitted constitutively by flowers or leaves of several plant species where it acts as an attractant for pollinators and as a chemical cue for host localization (or avoidance) for insects.^1–3 It is also released by some plant species after feeding damage inflicted by herbivorous insects and in this case might serve as a chemical cue for parasitoids to guide them to their prey (so called “indirect defense”).^4,5 Moreover, we have recently shown that plants can perceive cis-jasmone and that it acts as a signaling molecule in A. thaliana, inducing a discrete and distinctive suite of genes, of which a large subset is putatively involved in metabolism and defense responses.⁶ Cytochrome P450s feature prominently in these functional subsets and of these the highest fold change upon cis-jasmone treatment occurred with the cytochrome CYP81D11 (At3g28740).⁶ Hence this gene was chosen for a more thorough analysis of the potential biological relevance of the cis-jasmone induced defense response. Although the precise function of CYP81D11 remains to be determined, we could previously demonstrate its involvement in the indirect defense response in Arabidopsis, as plants exposed to cis-jasmone ceased to be attractive to the aphid parasitoid Aphidius ervi when this P450 was inactivated by T-DNA insertion mutagenesis.⁶ Here we report additional experiments which give further support to a role of CYP81D11 in the direct or indirect defense response of A. thaliana.Key words: cis-Jasmone, Cytochrome P450, indirect defense, tritrophic interactions, volatile signaling