Interactions of Avocado (Persea americana) Cytochrome P-450 with Monoterpenoids

The microsomal fraction of avocado (Persea americana) mesocarp is a rich source of cytochrome P-450 active in the demethylation of xenobiotics. Cytochrome P-450 from this tissue has been purified and well characterized at the molecular level (DP O'Keefe, KJ Leto [1989] Plant Physiol 89: 1141-1149; KR Bozak, H Yu, R Sirevag, RE Christoffersen [1990] Proc Natl Acad Sci USA 87: 3904-3908). Despite this extensive characterization, the role of the enzyme in vivo was not established. Optical and electron paramagnetic resonance binding studies described here suggest that the monoterpenoids, nerol and geraniol, are substrates of avocado cytochrome P-450 (spectral dissociation constant of 7.2 and 35 micromolar, respectively). Avocado microsomes have been shown to catalyze the hydroxylation of these monoterpenoids, and both nerol and geraniol have been shown to inhibit the activity of avocado cytochrome P-450 toward the artificial substrate 7-ethoxycoumarin, with nerol a competitive inhibitor of this activity.     

Cytochrome P-450 from the Mesocarp of Avocado (Persea americana)

The microsomal fraction from the mesocarp of avocado (Persea americana) is one of few identified rich sources of plant cytochrome P-450. Cytochrome P-450 from this tissue has been solubilized and purified. Enzymatic assays (p-chloro-N-methylaniline demethylase) and spectroscopic observations of substrate binding suggest a low spin form of the cytochrome, resembling that in the microsomal membrane, can be recovered. However, this preparation of native protein is a mixture of nearly equal proportions of two cytochrome P-450 polypeptides that have been resolved only under denaturing conditions. Overall similarities between these polypeptides include indistinguishable amino acid compositions, similar trypsin digest patterns, and cross reactivity with the same antibody. The amino terminal sequences of both polypeptides are identical, with the exception that one of them lacks a methionine residue at the amino terminus. This sequence exhibits some similarities with the membrane targeting signal found at the amino terminus of most mammalian cytochromes P-450.     

Acetyl-CoA Carboxylase Inhibitors from Avocado (Persea americana Mill) Fruits

A methanol extract of avocado fruits showed potent inhibitory activity against acetyl-CoA carboxylase, a key enzyme in fatty acid biosynthesis. The active principles were isolated and identified as (5E,12Z,15Z)-2-hydroxy-4-oxoheneicosa-5,12,15-trienyl (1), (2R,12Z,15Z)-2-hydroxy-4-oxoheneicosa-12,15-dienyl (2), (2R^*,4R^*)-2,4-dihydroxyheptadec-16-enyl (3) and (2R^*,4R^*)-2,4-dihydroxyheptadec-16-ynyl (4) acetates by instrumental analyses. The IC₅₀ of the compounds were 4.0×10^-6, 4.9×10^-6, 9.4×10^-6, and 5.1×10^-6M, respectively.     

Primary Structure of a Cysteine Proteinase Inhibitor from the Fruit of Avocado (Persea americana Mill)

The complete amino acid sequence of a proteinaceous cysteine proteinase inhibitor from the fruit of avocado (avocado cystatin) is presented. The protein consists of 100 amino acid residues and has a molecular mass of 11,300 Da. Comparison of this sequence with sequences of plant cysteine proteinase inhibitors (phytocystatins), including oryzacystatins I and II from rice seeds, cowpea cystatin, and corn cystatin, showed that the avocado cystatin molecule has 60% and 54% residues identical with the two forms of the rice seed proteins, oryzacystatins I and II, respectively, and 64% and 63% with the cowpea and corn proteins, respectively. The totally conserved sequence, Gln-Val-Val-Ala-Gly, among several of the animal cystatins as well as phytocystatins, is at positions 47-51 in the avocado cystatin molecule.     

Relationship between Fruit Softening and Wall Polysaccharides in Avocado (Persea americana Mill) Mesocarp Tissues

Changes in avocado (Persea americana) fruit texture during ripeningwere evaluated by stress-relaxation analysis. A conical probewas imposed into the mesocarp tissue to a depth of 0.6 mm andthe initial stress and the stress relaxation over 60 s weredetermined. The initial stress, an elastic parameter, was substantiallyreduced within one day when ripening was initiated by transferringthe fruit from 15 to 25°C. The minimum and maximum relaxationtime, parameters which reflect viscosity, were also reducedwithin one day. Mesocarp cell walls were fractionated into water-soluble(WS), hot EDTA-soluble (EDTA), alkaline soluble (hemicellulose)and the residual (cellulose) fractions. The amount of cellulosedid not change during ripening. Rhamnose, arabinose and uronicacids in the WS fraction increased during the initial day ofripening; those same components decreased in the EDTA fraction.A molecular weight downshift in the WS acidic polysaccharideswas detected within one day, while only slight changes wereobserved in the molecular weights of the EDTA fraction. Thequantities of individual sugar components of major hemicellulosefraction were unchanged, but there was a prominent molecularweight downshift in the xyloglucan components within one day.These results clearly revealed that both elastic and viscousproperties of avocado mesocarp tissues were substantially alteredduring ripening, and that the solubility changes in acidic polysaccharidesand decreases in the average molecular weight of cell wall xyloglucancomponents were associated with significant changes in fruittexture. (Received December 13, 1996; Accepted March 5, 1997)     

Pistil Starch Reserves at Anthesis Correlate with Final Flower Fate in Avocado (Persea americana)

A common observation in different plant species is a massive abscission of flowers and fruitlets even after adequate pollination, but little is known as to the reason for this drop. Previous research has shown the importance of nutritive reserves accumulated in the flower on fertilization success and initial fruit development but direct evidence has been elusive. Avocado (Persea americana) is an extreme case of a species with a very low fruit to flower ratio. In this work, the implications of starch content in the avocado flower on the subsequent fruit set are explored. Firstly, starch content in individual ovaries was analysed from two populations of flowers with a different fruit set capacity showing that the flowers from the population that resulted in a higher percentage of fruit set contained significantly more starch. Secondly, in a different set of flowers, the style of each flower was excised one day after pollination, once the pollen tubes had reached the base of the style, and individually fixed for starch content analysis under the microscope once the fate of its corresponding ovary (that remained in the tree) was known. A high variability in starch content in the style was found among flowers, with some flowers having starch content up to 1,000 times higher than others, and the flowers that successfully developed into fruits presented significantly higher starch content in the style at anthesis than those that abscised. The relationship between starch content in the ovary and the capacity of set of the flower together with the correlation found between the starch content in the style and the fate of the ovary support the hypothesis that the carbohydrate reserves accumulated in the flower at anthesis are related to subsequent abscission or retention of the developing fruit.     

On the Function of Seedcoats in Gibberellin-induced Hydrolysis of Starch Reserves during Avocado (Persea americana) Germination

Of all seed components investigated, the endogenous levels ofboth bound and free gibberellin (GA) are highest in embryo tissueof young immature avocado seeds. Experimentation with the gelatinaceousendosperm tissue of these seeds indicated that hydrolysis ofstarch reserves may be triggered by the embryo-contained GAacting on the ‘endosperm sheath’, a thin layer ofcells encompassing the endosperm. In the mature fruit when theendosperm and the encompassing layer are no longer present andin which starch reserves are present in the two stony cotyledons,the papery bi-integumental layer, commonly regarded as biologicallyinert, retains the ability to trigger starch hydrolysis uponcontact with GA. From these data and also based upon the findingsthat during germination levels of endogenous GA increase inembryo tissue and that exogenously applied GA₃ is effectivein induction of dormancy break it is proposed that in the avocadoseed an analogy, physiological but not anatomical, to the cerealendosperm system exists.     

First Report of Root Rot Caused by Ilyonectria (=Neonectria) macrodidyma on Avocado (Persea americana) in Italy

During the 2009 and the 2010 growing seasons, a root rot disease has been detected on young potted Persea americana plants in two nurseries located in the Catania and Messina provinces (eastern Sicily, Italy). A Cylindrocarpon sp. was consistently recovered from pieces of symptomatic tissues on Petri dishes containing potato dextrose agar. On the basis of morphological characteristics and molecular identification by DNA sequencing and phylogenetic analysis of internal transcribed spacer and β‐tubulin gene regions, the causal agent was identified as Ilyonectria (=Neonectria) macrodidyma. Koch’s postulates were fulfilled by pathogenicity tests carried out on potted P. americana seedlings. To our knowledge, this is the first to report worldwide of the occurrence of a disease caused by I. macrodidyma on P. americana.     

Cytochrome P-450-catalysed monoterpenoid oxidation in catmint (Nepeta racemosa) and avocado (Persea americana); evidence for related enzymes with different activities

A cytochrome P-450 present in ripening avocado (Persea americana) fruit mesocarp (CYTP71A1) had previously been shown to metabolize the monoterpenoids nerol and geraniol (Hallahan et al. (1992) Plant Physiol. 98, 1290-1297). Using DNA encoding CYP71A1 as a hybridization probe, we have shown by Southern analysis that a related gene is present in the catmint, Nepeta racemosa. RNA blot analysis, together with Western analysis of catmint leaf polypeptides using avocado cyt P-450 antiserum, showed that a closely related gene is expressed in catmint leaves. Cytochrome P-450 in catmint microsomes catalysed the specific hydroxylation of nerol and geraniol at C-10, whereas avocado CYP71A1, in either avocado microsomes or heterologously expressed in yeast, catalysed 2,3- or 6,7-epoxidation of these substrates. These results suggest that orthologous genes of the CYP71 family are expressed in these two plant species, but catalyse dissimilar reactions with monoterpenoid substrates.     

植物细胞色素P450基因的异源表达系统研究进展

细胞色素P450氧化酶是一类具有多种催化功能含血红素的氧化酶系。由于参与多种类型的氧化反应,在植物生命活动中有重要功能,其研究一直受到重视。自1990年第一个植物P450基因成功克隆以来,到2002年年底,已有600多个P450基因被克隆,有100多个基因在细菌、酵母、杆状病毒昆虫细胞等异源表达系统中成功表达并鉴定了功能。拟对植物P450的大肠杆菌、酵母、杆状病毒昆虫细胞表达系统的特点进行比较,对在各表达系统中成功表达并进行了功能鉴定的植物P450进行归纳,并对目前植物P450异源表达的现状和应用进行概述。     

Cytochrome P450 reductase (POR)as a ferroptosis fuel

Oxygen,iron,and polyunsaturated fatty acids (PUFAs;fatty acids containing more than one double bond) are all bene-ficial to our cellular lives.Incorporation of these components into cellular processes,however,comes at a cost:the bis-allylic structure of PUFAs and the enrichment of cellular environments with iron and oxygen render PUFA-containing phospholipids (PUFA-PLs) particularly susceptible to per-oxidation (Yang and Stockwell,2016).Accumulation of lethal amounts of lipid peroxides in cell membranes leads to a form of cell death known as ferroptosis (Dixon et al.,2012;Stockwell et al.,2017;Stockwell and Jiang,2020).Conse-quently,cells are equipped with strong antioxidant defense systems that constantly dissipate toxic lipid peroxides gen-erated in cellular membranes,thereby maintaining cell via-bility and homeostasis (Zheng and Conrad,2020).The most powerful anti-ferroptosis defense system is believed to be mediated by glutathione peroxidase 4 (GPX4),a glutathione peroxidase that uses glutathione as its cofactor to reduce lipid hydroperoxides to non-toxic lipid alcohols (Fig.1)(Zheng and Conrad,2020).A variety of ferroptosis inducers(FINs) act to inactivate GPX4 or deplete glutathione,causing an imbalance between the production and detoxification of lipid peroxides that subsequently induces ferroptotic cell death (Yang et al.,2014).Genetic ablation of GPX4 can have the same effect (Friedmann Angeli et al.,2014).     

Polyuronides in Avocado (Persea americana) and Tomato (Lycopersicon esculentum) Fruits Exhibit Markedly Different Patterns of Molecular Weight Downshifts during Ripening

Avocado (Persea americana) fruit experience a rapid and extensive loss of firmness during ripening. In this study, we examined whether the chelator solubility and molecular weight of avocado polyuronides paralleled the accumulation of polygalacturonase (PG) activity and loss in fruit firmness. Polyuronides were derived from ethanolic precipitates of avocado mesocarp prepared using a procedure to rapidly inactivate endogenous enzymes. During ripening, chelator (cyclohexane-trans-1,2-diamine tetraacetic acid [CDTA])-soluble polyuronides increased from approximately 30 to 40 [mu]g of galacturonic acid equivalents (mg alcohol-insoluble solids)-1 in preripe fruit to 150 to 170 [mu]g mg-1 in postclimacteric fruit. In preripe fruit, chelator-extractable polyuronides were of high molecular weight and were partially excluded from Sepharose CL- 2B-300 gel filtration media. Avocado polyuronides exhibited marked downshifts in molecular weight during ripening. At the postclimacteric stage, nearly all chelator-extractable polyuronides, which constituted from 75 to 90% of total cell wall uronic acid content, eluted near the total volume of the filtration media. Rechromatography of low molecular weight polyuronides on Bio-Gel P-4 disclosed that oligomeric uronic acids are produced in vivo during avocado ripening. The gel filtration behavior and pattern of depolymerization of avocado polyuronides were not influenced by the polyuronide extraction protocol (imidazole versus CDTA) or by chromatographic conditions designed to minimize interpolymeric aggregation. Polyuronides from ripening tomato (Lycopersicon esculentum) fruit extracted and chromatographed under conditions identical with those used for avocado polyuronides exhibited markedly less rapid and less extensive downshifts in molecular weight during the transition from mature-green to fully ripe. Even during a 9-d period beyond the fully ripe stage, tomato fruit polyuronides exhibited limited additional depolymerization and did not include oligomeric species. A comparison of the data for the avocado and tomato fruit indicates that downshifts in polyuronide molecular weight are a prominent feature of avocado ripening and may also explain why molecular down-regulation of PG (EC 3.2.1.15) in tomato fruit has resulted in minimal effects on fruit performance until the terminal stages of ripening.     

Avocado seeds (Persea americana cv. Criollo sp.): Lipophilic compounds profile and biological activities

This study aimed to evaluate the volatile or lipophilic chemical profiling and the biological activities of avocado (Persea americana cv. Criollo sp.) seed extracts. Chemical profile of volatile compounds (GC/MS), antioxidant properties (phenolic compounds, DPPH radical scavenging activities and reducing power), and antimicrobial activity (Salmonella Typhimurium and Staphylococcus aureus) of avocado (Persea americana cv. Criollo sp.) seed extracts (ethanol and acetone) were characterized. Sixteen volatile chemical compounds were determined, including isoprenoid derivatives (estragole), esters of fatty acids (linoleic and linolenic acids), and their derivatives (9,12-Octadecadien-1-ol and 9,12,15-Octadecatrien-1-ol). Acetone was the best solvent to obtain volatile compounds from avocado seed; this extract also showed a higher reducing power (56.35 mg AAE/100 g). Maximum S. aureus and S. Typhimurium log reductions were 4.0 ± 0.3 and 1.8 ± 0.3 at the highest amount used (2000 mg/L), without significant effect (p < 0.05) of the solvent used. According to the results of the volatile chemical profiling of avocado (Persea americana cv. Criollo sp.) seed extracts, they can have potential application as antioxidant (212.75 and 183.75 mg Trolox/100 g) and antimicrobial additives.     

Cytochrome P450 (P450) isoenzyme specific dealkylation of alkoxyresorufins in rat brain microsomes

Characterization of xenobiotic metabolizing cytochrome P450s (P450s) was carried out in rat brain microsomes using the specific substrates, 7-pentoxy- and 7-ethoxyresorufin (PR and ER), metabolized in the liver by P450 2B1/2B2 and 1A1/1A2 respectively and 7-benzyloxyresorufin (BR), a substrate for both the isoenzymes. Brain microsomes catalysed the O-dealkylation of PR, BR and ER in the presence of NADPH. The ability to dealkylate alkoxyresorufins varied in different regions of the brain. Microsomes from the olfactory lobes exhibited maximum pentoxyresorufin-O-dealkylase (PROD), benzyloxyresorufin-O-dealkylase (BROD) and ethoxyresorufin-O-dealkylase (EROD) activities. The dealkylation was found to be inducer selective. While pretreatment with phenobarbital (PB; 80 mg/kg; i.p. × 5 days) resulted in significant induction in PROD (3-4 fold) and BROD (4-5 fold) activities, 3-methylcholanthrene (MC; 30 mg/kg; i.p. × 5 days) had no effect on the activity of PROD and only a slight effect on that of BROD (1.4 fold). MC pretreatment significantly induced the activity of EROD (3 fold) while PB had no effect on it. Kinetic studies have shown that this increase in the activities following pretreatment with P450 inducers was associated with a significant increase in the velocity of the reaction (V_max) of O-dealkylation. In vitro studies using organic inhibitors and antibodies have further provided evidence that the O-dealkylation of alkoxyresorufins is isoenzyme specific. While in vitro addition of a-naphthoflavone (ANF), an inhibitor of P450 1A1/1A2 catalysed reactions and antibody for hepatic P450 1A1/1A2 isoenzymes produced a concentration-dependent inhibition of EROD activity, metyrapone, an inhibitor of P450 2B1/2B2 and antibody for hepatic P450 2B1/2B2 significantly inhibited the activity of PROD and BROD in vitro. The data suggest that, as in the case of liver, dealkylation of alkoxyresorufins can be used as a biochemical tool to characterise the xenobiotic metabolising P450s and substrate selectivity of P450 isoenzymes in rat brain microsomes.     

Recombinant Cytochrome P-450 Production in Yeast

Lipid membranes are versatile and convenient alternatives to study the properties of natural cell membranes. Self-assembled, artificial, substrate-supported lipid membranes have taken a central role in membrane research due to a combination of factors problem are seemingly incompatible with each other. For example, methods to produce spacers which give suitable wetting conditions of the substrate will be more compatible with for example spotting arrays than other patterning methods. And it is likely that spreading of membranes on polymer cushions opens up new ways of creating artificial replicas of complex natural membranes which have been challenging on solid supports, as it already seems that tethered membranes can help prepare and improve stability of, for example, aperture spanning membranes. The merger of these approaches has only just started.

In summary, SLBs combine added stability, defined localization and application of surface sensitive and on the nanoscale localized measurement techniques, which ensures that they will continue to attract much attention and research effort in the years to come. And while there is no shortage of remaining scientific challenges in the field, there is also no lack of creative solutions being developed to meet these challenges.     

Genetic relationships within avocado (Persea americana Mill) cultivars and between Persea species

The origin of avocado (Persea americana Mill) cultivars, as well as the genetic relationships between Persea species, are not well defined and are based mainly on morphological parameters. Minisatellite DNA markers were used to analyze 24 P. americana cultivars in an attempt to define their racial allocation. DNA mixes representing the three races were evaluated and used for analysis. The allocation of 19 of the above cultivars was substantiated by the DNA markers, while new suggestions were offered regarding the remaining five. Eight cultivars, of unknown racial origin, were also examined, and a phylogenetic tree suggesting their origin is offered. Selfing progeny of five cultivars were analyzed for six morphological traits which differentiate the three races, and were compared to their parents in order to assess their origin. Eleven Persea species were analyzed, using DNA fingerprint patterns and SSR (simple sequence repeat) alleles, in order to identify the genetic relationships among the Persea species, and between them and the three P. americana races. The phylogenetic tree obtained is presented. The high value of variation between the avocado cultivars and Persea species observed in this work, suggests that the validity of race and species definition within Persea be treated with caution. Received: 3 August 1996 / Accepted: 23 August 1996     

人细胞色素P450在大肠杆菌中的功能表达

研究人细胞色素P450（P450,CYP）在大肠杆菌中的功能表达对新药研发,临床药物治疗和药物早期ADME/T性质研究均有重要意义。异源表达人P450使用最多的宿主是大肠杆菌E.coli,然而要获得足量的有催化活性的P450仍是一个难题。结合作者近年研究,对异源表达的研究意义,P450在E.coli中功能表达的策略,高效表达的影响因素和共表达等方面做一评述,指出今后的研究应用方向。