Influence of avocado (Persea americana) Cx-cellulase on the structural features of avocado cellulose

Avocado (Persea americana Mill.) fruit produce copious quantities of the enzyme Cx-cellulase (EC 3.2.1.4) during ripening. The possibility that Cx-cellulase is able to disrupt cellulose microfibril oranization was investigated using molecular weight (M_r), x-ray diffraction, and ultrastructural analyses of cell walls from unripe avocado fruit incubated with the purified enzyme. Results indicate that Cx-cellulase causes a downshift in the M_r of unbranched cell-wall polymers in the M_r range of 10⁶–10⁷ Da. There is an increase in the proportion of crystalline cellulose, and cellulose fibrils appear to lose cohesiveness in response to enzyme activity. We propose that Cx-cellulase attacks avocado cellulose at accessible sites in the peripheral and integral noncrystalline regions of the microfibril, resulting in a loss of cohesiveness within the fibril structure and an alteration in the binding of associated cell-wall matrix polysaccharides. The initial loss of avocado mesocarp firmness during fruit ripening may be linked to the onset of Cx-cellulase activity.Abbreviations CMC carboxymethylcellulose - DMAC dimethylacetamide - DS developmental stage - M molecular weight - XG xyloglucan     

N-demethylation of p-chloro-N-methylaniline catalyzed by subcellular fractions from the avocado pear (Persea americana)

Subcellular fractions from the avocado pear ( Persea americana) catalyzed formation of p-chloroaniline from p-chloro-N-methylaniline. Fractions prepared by centrifugation of avocado homogenates at 20, 000g for 20 min formed p-chloroaniline (2900 +/- 500 pmol min-1 mg protein-1) with an NADPH-generating system. p-Chloroaniline formation required reduced pyridine nucleotide (NADPH was 6-7 times more effective than NADH) and O2. N-Demethylation was inhibited by CO (55% inhibition at CO:O2 = 1) and was not inhibited by CN. Cytochrome P-450 was detected in the 20, 000g pellet at levels of 300-380 pmol/mg protein. This particulate preparation was also active in catalyzing the NADPH-dependent epoxidation of the chlorinated cyclodiene aldrin. Improvements to a colorimetric procedure for measuring p-chloroaniline increased the sensitivity of the procedure fourfold, and allowed use of samples containing high amounts of lipid. Avocado pear is suitable tissue for further studies on the oxidation of foreign compounds by higher plants.     

Regulation of defence responses in avocado roots infected with Phytophthora cinnamomi (Rands)

Phytophthora cinnamomi occurs worldwide and has a host range in excess of 1,000 plant species. Avocados (Persea americana Mill) have been described as highly susceptible to this soil-borne pathogen. Here, the regulation of defence responses in avocado root seedlings inoculated with P. cinnamomi mycelia is described. A burst of reactive oxygen species (ROS) was observed 4 days after inoculation. The higher physiological concentration of H₂O₂ induced by P. cinnamomi on avocado roots had no effect on in vitro growth of the oomycete. Total phenols and epicathecin content showed a significant decrease, but lignin and pyocianidins exhibited no changes after inoculation. Also, increased nitric oxide (NO) production was observed 72 h after treatment. We studied the effects of one NO donor [sodium nitroprusside (SNP)], and one NO scavenger [2- to 4-carboxyphenyl-4,4,5,5-tetramethylimidazole-1-oxyl-3-oxide (CPTIO)] to determine the role of NO during root colonisation by P. cinnamomi mycelia. Pretreatment of the roots with CPTIO, but not with SNP, inhibited root colonisation suggesting an important role for NO production during the avocado–P. cinnamomi interaction. Our data suggest that although defence responses are activated in avocado roots in response to P. cinnamomi infection, these are not sufficient to avoid pathogen invasion.     

Some aspects of carnitine metabolism in avocado (Persea americana) (Short Communication)

The fact that palmitoyl-l-carnitine is oxidized by avocado mitochondria at a rate comparable with that of succinate oxidation suggests that there are at least two systems for β-oxidation in higher plants. The carnitine-associated system is located in a mitochondrial fraction, whereas the glyoxylate-cycle-associated system is located in the glyoxysomes.     

Differential selection pressure exerted by root rot disease on the microbial communities in the rhizosphere of avocado (Persea americana Mill.)

Despite the high economic impact of root rot disease, knowledge regarding the rhizosphere microflora of avocado trees affected by root rot is limited. Metagenomics was applied to identify the difference in the rhizosphere microflora of avocado trees with and without visible symptoms of root rot. Approximately, 446,970 common gene catalogues differed between them, confirming that root rot affected the bacterial and fungal communities in the rhizosphere. The proportion of bacterial genera, namely, Labilithrix, Sorangium, Sandaracinus and Pedosphaera showed a decrease while Phenylobacterium, Rhizomicrobium, Candidatus Solibacter and Silvibacterium genera were increased by root rot. The proportion of fungal genera, namely, Pseudogymnoascus, Moelleriella, Mortierella, Lepidopterella, Babjeviella, Lachancea, Macrophomina, Pneumocystis, Sugiyamaella and Cyphellophora showed an increase while Cryptococcus, Verticillium, Bipolaris, Pyrenochaeta, Rhizophagus, Cenococcum and Neonectria genera were inhibited by root rot. Moreover, the proportion of the top 10 bacteria in the rhizosphere of symptomatic trees was significantly higher, and that of the top 10 fungi was significantly lower, compared to the asymptomatic trees. Principal component analysis based on abundance analysis and function prediction showed that in symptomatic trees, the bacterial community was more concentrated, while the fungal community was more dispersed. The differences in the responses of bacterial and fungal genera suggested that the pathogenic fungi exert varying selection pressure on the microflora. Moreover, root rot affected the metabolism of carbohydrates, lipids and amino acids in bacteria, and the global and overview maps, carbon metabolism and processing of genetic and environmental information in fungi, which might result in differential selection pressure.     

Purification and catalytic properties of polygalacturonase isoforms from ripe avocado (Persea americana) fruit mesocarp

Endo-polygalacturonase (PG; EC 3.2.1.15) was recovered from the cell walls of avocado mesocarp ( Persea americana Mill cv. Lula) tissue and purified by sequential ion exchange and gel permeation chromatography. Two isoforms (S-I and S-II) were recovered, exhibiting molecular masses of about 41 kD on size exclusion media and about 48 (S-I) and 46 (S-II) kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both isoforms exhibited maximum activity at pH 6.0 against polygalacturonic acid (PGA) and hydrolyzed PGA of about 180 kDa to polymers of about 4 kDa. The catalytic activity of the 48-kDa isoform against PGA was slightly higher than that of the 46-kDa isoform. The purified PGs catalyzed significant molecular mass downshifts in the polyuronides of pre-ripe avocados; however, the capacity of the enzymes to solubilize polyuronides from cell walls of pre-ripe fruit was limited.     

Evidence for the presence of a native,non-auxinic rooting promoter in avocado (Persea americana Mill.)

The presence of a rooting promoter in paratially purified extracts of avocado (Persea amricana Mill.) organs has been demonstrated using the mung bean rooting bioassay. Extraction with 80% methanol was followed by partition into diethyl ether, paper chromatography (PC) and 3 steps of thin layer chromatography (TLC). The number of roots induced by the rooting promoter in the absence of exogenous auxin was 5 to 7 times higher than that of the water control and 50% higher than by 4-(indol-3-yl) butyric acid (IBA) at its reported optimum concentration. Rooting of tomato, Coleus and young avocado cuttings was also enhanced by the rooting promoter. The rooting promoter was inhibitory in the wheat coleoptile section elongation bioassay for auxins and had slight inhibitory activity in the split pea stem curvature test.The biological properties of the avocado rooting promoter may be comparable to those of -(p-chlorophenoxy) isobutyric acid (PCIB) which acts as an anti-auxin in certain bioassays and, nevertheless, promotes the rooting of mung bean cuttings.     

Enhancing frequency of regeneration of somatic embryos of avocado (Persea americana Mill.) using semi-permeable cellulose acetate membranes

Regeneration of avocado via somatic embryogenesis is difficult due to poor embryo maturation, resulting in low frequencies of germination. In this study, the influence of semi-permeable cellulose acetate membranes and culture media, containing high levels of sucrose along with coconut water, on maturation and germination of somatic embryos of avocado have been evaluated. The culture of embryogenic calli on top of cellulose acetate membranes significantly increased the number of mature, white-opaque embryos that were recovered after 5 weeks of culture. These embryos showed a much more normal appearance and better quality compared with the control embryos, although the embryo size was significantly reduced. To increase the embryo size and to complete maturation, several two-step maturation treatments were tested. The culture of white-opaque somatic embryos in a modified MS medium with B5 macronutrients gelled with 10 g L^?1 agar (B5m10A medium) over a 5-week period, followed by 5 additional weeks in B5m10A with 45 g L^?1 sucrose and 20 % coconut water, yielded the best results, reducing the percentage of necrotic embryos and the number of calli formed. The beneficial effects of this maturation treatment were enhanced when using embryos that were pre-matured on cellulose acetate membranes. Following this two-step maturation treatment, the germination rate of the control somatic embryos, which were not cultured on cellulose membranes, was lower than 10 %, but it significantly improved when the embryos had been pre-matured on cellulose acetate membranes for 5 weeks, reaching a germination rate close to 40 %. The water availability was significantly reduced when somatic embryos were cultured on cellulose membranes, and after this pre-maturation treatment, the white-opaque embryos showed lower water potential and ABA content compared with the control embryos. These results suggest that culturing over cellulose membranes causes a controlled embryo desiccation that enhances the recovery of plants.     

Identification of major sporulation proteins of Myxococcus xanthus using a proteomic approach

Myxococcus xanthus is a soil-dwelling, gram-negative bacterium that during nutrient deprivation is capable of undergoing morphogenesis from a vegetative rod to a spherical, stress-resistant spore inside a domed-shaped, multicellular fruiting body. To identify proteins required for building stress-resistant M. xanthus spores, we compared the proteome of liquid-grown vegetative cells with the proteome of mature fruiting body spores. Two proteins, protein S and protein S1, were differentially expressed in spores, as has been reported previously. In addition, we identified three previously uncharacterized proteins that are differentially expressed in spores and that exhibit no homology to known proteins. The genes encoding these three novel major spore proteins (mspA, mspB, and mspC) were inactivated by insertion mutagenesis, and the development of the resulting mutant strains was characterized. All three mutants were capable of aggregating, but for two of the strains the resulting fruiting bodies remained flattened mounds of cells. The most pronounced structural defect of spores produced by all three mutants was an altered cortex layer. We found that mspA and mspB mutant spores were more sensitive specifically to heat and sodium dodecyl sulfate than wild-type spores, while mspC mutant spores were more sensitive to all stress treatments examined. Hence, the products of mspA, mspB, and mspC play significant roles in morphogenesis of M. xanthus spores and in the ability of spores to survive environmental stress.     

Characterization and evolutionary analysis of a large polygalacturonase gene family in the oomycete plant pathogen Phytophthora cinnamomi

Polygalacturonases (PGs) are secreted by fungal pathogens during saprophytic and parasitic growth, and their degradation of pectin in the plant cell wall is believed to play a major role in tissue invasion and maceration. In this study, PG activity was demonstrated in culture filtrates of the oomycete plant pathogen, Phytophthora cinnamomi. A P. cinnamomi pg gene fragment amplified using degenerate primers based on conserved regions in fungal and plant PGs was used to isolate 17 complete P. cinnamomi pg genes and pseudogenes from a genomic library and partial sequence for another two genes. Gel blotting of genomic DNA indicated that there may be even more pg genes in the P. cinnamomi genome. P. cinnamomi pg gene sequences were expressed in PG-deficient yeast and found to confer PG activity, thereby confirming their functional identity. The predicted mature P. cinnamomi PGs fall into subgroups that exhibit large differences in the extent of N-glycosylation, isoelectric points, and N- and C-terminal structure. Evidence for birth-and-death and reticulate evolution in the P. cinnamomi pg gene family was obtained, and some codons for surface exposed residues in the P. cinnamomi PGs were shown to have been subject to diversifying selection. Contrary to accepted phylogenies for other proteins, phylogenetic analysis of the P. cinnamomi PGs revealed a closer relationship with PGs from true fungi than with those from plants.     

In vivo anticoccidial,antioxidant, and anti-inflammatory activities of avocado fruit,Persea americana (Lauraceae), against Eimeria papillata infection

Apicomplexan parasites, especially Eimeria sp., are the main intestinal murine pathogens, that lead to severe injuries to farm and domestic animals. Many anticoccidial drugs are available for coccidiosis, which, leads to the development of drug-resistant parasites. Recently, natural products are considered as an alternative agent to control coccidiosis. This study was designed to evaluate the anticoccidial activity of the Persea americana fruit extract (PAFE) in male C57BL/6 mice. A total of 35 male mice were divided into seven equal groups (1, 2, 3, 4, 5, 6, and 7). At day 0, all groups except the first group which served as uninfected-untreated control were infected orally with 1 × 10³ E. papillata sporulated oocysts. Group 2 served as uninfected-treated control. Group 3 was considered an infected-untreated group. After 60 min of infection, groups 4, 5, and 6 were treated with oral doses of PAFE aqueous methanolic extract (100, 300, and 500 mg/kg of body weight, respectively). Group 7 was treated with amprolium (a reference drug for coccidiosis). PAFE with 500 mg/kg, was the most effective dose, inducing a significant reduction in the output of oocysts in mice feces (by about 85.41%), accompanied by a significant decrease in the number of the developmental parasite stages and a significant elevation of the goblet cells in the jejunal tissues. Upon treatment, a significant change in the oxidative status due to E. papillata infection was observed, where the levels of glutathione (GSH) increased, while, levels of malondialdehyde (MDA) and nitric oxide (NO) were decreased. In addition, the infection significantly upregulated the inflammatory cytokines of interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α), and interferon-γ (IFN-γ). This increase in mRNA expression of IL-1β, TNF-α, and IFN-γ was about 8.3, 10.6, and 4.5-fold, respectively, which significantly downregulated upon treatment. Collectively, P. americana is a promising medicinal plant with anticoccidial, antioxidant, and anti-inflammatory activities and could be used for the treatment of coccidiosis.     

Identification of disease-associated proteins by proteomic approach in ankylosing spondylitis

Ankylosing spondylitis (AS) is a chronic systemic inflammatory disorder of the axial skeleton and shows significant inherited susceptibility. Auto-immune responses have been traditionally considered as a putative pathogenetic event in AS. However, no consistent self-antigen has been identified to responsible for the disorders in AS to this day. In this study, serum protein profiles of AS patients and healthy controls from a large Chinese AS family were investigated by two dimensional electrophoresis analysis. A group of four highly expressed protein spots was observed in all AS patients' profiles and subsequently identified as isoforms of haptoglobin precursor (pre-Hp) by ESI-Q-TOF MS/MS. Increased expression of haptoglobin precursor were also observed in sera of sporadic AS patients. Moreover, bioinformatics analysis revealed epitopes derived from haptoglobin precursor with high affinity binding to HLA-B( *)2705, a primary subtype associated with AS. These results indicate that pre-Hp may be involved in the pathogenesis of AS.     

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.     

Integrating gene flow, crop biology, and farm management in on-farm conservation of avocado (Persea americana, Lauraceae)

Maintaining crop diversity on farms where cultivars can evolve is a conservation goal, but few tools are available to assess the long-term maintenance of genetic diversity on farms. One important issue for on-farm conservation is gene flow from crops with a narrow genetic base into related populations that are genetically diverse. In a case study of avocado (Persea americana var. americana) in one of its centers of diversity (San Jerónimo, Costa Rica), we used 10 DNA microsatellite markers in a parentage analysis to estimate gene flow from commercialized varieties into a traditional crop population. Five commercialized genotypes comprised nearly 40% of orchard trees, but they contributed only about 14.5% of the gametes to the youngest cohort of trees. Although commercialized varieties and the diverse population were often planted on the same farm, planting patterns appeared to keep the two types of trees separated on small scales, possibly explaining the limited gene flow. In a simulation that combined gene flow estimates, crop biology, and graft tree management, loss of allelic diversity was less than 10% over 150 yr, and selection was effective in retaining desirable alleles in the diverse subpopulation. Simulations also showed that, in addition to gene flow, managing the genetic makeup and life history traits of the invasive commercialized varieties could have a significant impact on genetic diversity in the target population. The results support the feasibility of on-farm crop conservation, but simulations also showed that higher levels of gene flow could lead to severe losses of genetic diversity even if farmers continue to plant diverse varieties.     

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.     

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.     

Peroxidase activities and isoenzyme profiles associated with development of avocado (Persea americana,M.) leaves at different ontogenetic stages

Changes in four peroxidase activity fractions (soluble, membrane-bound, as well as ionically and covalently bound) were studied during development of juvenile and adult avocado leaves. Greater differences were found in the soluble fraction with an increase in total activity at the end of the growth phase. In relation to the ontogenetic stages, there were significant variations in the soluble peroxidase activity of both stages, especially in leaves which have already detained their growth, 263 U/g fresh wt in adult leaves vs. 70 U/g fresh wt juvenile leaves. Moreover, the isozyme profile of this fraction revealed the appearance of an anionic band, R_f 0.35, at much earlier stages in juvenile than in adult leaves. Concerning the other three fractions, there were no marked changes in total activity of either membrane-bound or ionically and covalently bound peroxidases. However, in the isoenzyme profiles of the ionically bound fraction of juvenile leaves, three highly cationic bands appeared at much earlier stages than in adult leaves. In avocado, attempts to use leaf peroxidase activity as marker of ontogenetic age must be taken with caution, since great fluctuations related with developmental stages occur in juvenile and adult leaves.