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 x 10(-6), 4.9 x 10(-6), 9.4 x 10(-6), and 5.1 x 10(-6) M, respectively.     

An EST-SSR-based linkage map for Persea americana Mill. (avocado)

Recent enhancement of the pool of known molecular markers for avocado has allowed the construction of the first moderately dense genetic map for this species. Over 300 SSR markers have been characterized and 163 of these were used to construct a map from the reciprocal cross of two Florida cultivars 'Simmonds' and 'Tonnage'. One hundred thirty-five primer pairs amplified 163 usable loci with 20 primer pairs amplifying more than one locus. 'Tonnage' was heterozygous for 152 (93%) loci, whereas 'Simmonds' was heterozygous for 64 (39%). Null alleles were identified at several loci. Linkage maps were produced for both reciprocal crosses and combined to generate a composite linkage map for the F₁ population of 715 individuals. The composite map contains 12 linkage groups. Linkage groups ranged in size from 157.3 cM (LG2) to 2.4 cM (LG12) and the number of loci mapped per group ranged from 29 (LG1) to two (LG12). The total map length was 1,087.4 cM. Only seven markers were observed to have segregation distortion (α ≤ 0.05) across both sub-composite (reciprocal) maps. Phenotypic data from traits of horticultural interest are currently being collected on this population with the ultimate goal of identifying useful quantitative trait loci and the development of a marker-assisted selection program.     

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     

Seventy microsatellite markers from Persea americana Miller (avocado) expressed sequence tags

Expressed sequence tags for Persea americana Mill. were investigated to expand upon the number of informative microsatellite markers available for avocado. Seventy informative loci were discovered using 24 P. americana var. americana Mill. accessions. The number of alleles detected ranged from two to 17 and averaged 7.1 alleles per locus. These primers successfully amplified products in different varieties of P. americana, hybrids and a related species, Persea schiedeana. These primers will be useful for characterizing germplasm, determining genetic relationships of cultivated accessions, and for marker‐assisted development of root rot‐tolerant P. americana var. americana rootstock material.     

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     

Two glucosylated abscisic acid derivates from avocado seeds (Persea americana Mill. Lauraceae cv. Hass)

Phytochemical investigation of avocado seed material (Persea americana Mill., Lauraceae) resulted in the isolation of two glucosylated abscisic acid derivates. One of these was not known as a natural product and can be regarded as a potential 'missing link' in abscisic acid metabolism in plants. After fractionation by high-speed countercurrent chromatography, and multiple steps of column chromatography, structures were elucidated by 1D-, 2D-NMR, electrospray-MS to be the novel beta-d-glucoside of (1'S,6'R)-8'-hydroxyabscisic acid, and (1'R,3'R,5'R,8'S)-epi-dihydrophaseic acid beta-d-glucoside. Absolute configuration was determined by circulardichroism, optical rotation, and by NOE experiments.     

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.     

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.     

Somatic embryogenesis in avocado (Persea americana Mill.) in vitro

A tissue culture procedure was developed for the regeneration of somatic embryos from callus cultures of the avocado,Persea americana. Immature zygotic embryos, 0.6–0.8 mm long, were used as original explants. Addition of 0.1 mg/l picloram (4-amino-3,5,6-trichloropicolinic acid) to culture medium appeared critical for callus initiation. Development of somatic embryos was accomplished in picloram concentrations of 0.01 to 0.1 ml/l. A few well developed embryos produced green shoots. Attempts to induce a higher incidence of germination were unsuccessful.Investigation was supported by funds from the California Avocado Society, the Instituto Nacional de Investigaciones Agrarias, the Fundation Juan March, the Chancellor's Patent Fund, and the Southern California Phi Beta Kappa Alumni Association. Authors thank H. Quick for the photography and J. Lippert for the illustrations.     

Cytokinin and inhibitor activities in the avocado fruit mesocarp

No cytokinin activity was found in methanolic mesocarp extracts after purification with petroleum ether and ethyl acetate. Cytokinin activity did appear, however, in aqueous fraction after acid hydrolysis or passage through Dowex 50 (H⁺) ion exchange columns. The level of this activity, the fruit growth rate, and the cell division rate were found to be positively correlated (with each other).     

Microsatellite markers in avocado (Persea americana Mill.): genealogical relationships among cultivated avocado genotypes

Twenty-five microsatellite markers uniquely differentiated 35 avocado cultivars and two wild relatives. Average heterozygosity was high (60.7%), ranging from 32% in P. steyermarkii to 84% in Fuerte and Bacon. In a subset of 15 cultivars, heterozygosity averaged 63.5% for microsatellites, compared to 41.8% for restriction fragment length polymorphisms (RFLPs). A neighbor-joining tree, according to average shared allele distances, consisted of three clusters likely corresponding to the botanical races of avocado and intermediate clusters uniting genotypes of presumably racially hybrid origin. Several results were at odds with existing botanical assignments that are sometimes rendered difficult by incomplete pedigree information, the complexity of the hybrid status (multiple backcrossing), or both. For example, cv. Harvest clustered with the Guatemalan race cultivars, yet it is derived from the Guatemalan x Mexican hybrid cv. Gwen. Persea schiedeana grouped with cv. Bacon. The rootstock G875 emerged as the most divergent genotype in our data set. Considerable diversity was found particularly among accessions from Guatemala, including G810 (West Indian race), G6 (Mexican race), G755A (hybrid Guatemalan x P. schiedeana), and G875 (probably not P. americana). Low bootstrap support, even upon exclusion of (known) hybrid genotypes from the data matrix, suggests the existence of ancient hybridization or that the botanical races originated more recently than previously thought.     

Isolation, culture and regeneration of avocado (Persea americana Mill.) protoplasts

Protoplasts were isolated from embryogenic suspension cultures derived from avocado (Persea americana Mill.) zygotic embryos and nucellus in an enzyme digestion solution consisting of 1% cellulase Onozuka RS, 1% Macerase R10, 0.2% Pectolyase Y-23, 0.7 M mannitol. 24.5 mM CaCl₂, 0.92 mM NaH₂PO₄ and 6.25 2-[N-morpholino]ethanesulfonic acid (1.5 ml) mixed with 0.7 M MS^–8P (2.5 ml). MS^-8P medium consisted of Murashige and Skoog salts without NH₄NO₃, 1 mg l^–1 thiamine HCl, 100 mg l^–1 myo-inositol, 3.1 g l^–1 glutamine and 8P organic addenda. Medium osmolarity was adjusted with 0.15 M sucrose and 0–0.55 M mannitol. Protoplast yields of 3.5×10⁶ protoplasts g^–1 were obtained. Growth and development of the protoplasts were significantly affected by osmolarity, nitrogen source, plating density and culture medium dilution. Under optimum conditions, proembryos developed directly from embryogenic protoplasts and subsequently into somatic embryos. Optimum conditions for somatic embryo development included the culture of protoplasts at a density of 0.8–1.6×10⁵ ml^–1 in 0.4 M MS^–8P for 2–3 weeks, followed by subculture in 0.15 M MS^–8P at a diluted density of 20–40× for 1 month in darkness to obtain somatic embryos. Mature somatic embryos were recovered on semisolid medium; however, a low frequency of plantlet recovery (≤1%) from protoplast-derived somatic embryos was observed. Received: 9 February 1998 / Revision received: 4 May 1998 / Accepted: 15 May 1998     

Nucleotide diversity and linkage disequilibrium in wild avocado (Persea americana Mill.)

Resequencing studies provide the ultimate resolution of genetic diversity because they identify all mutations in a gene that are present within the sampled individuals. We report a resequencing study of Persea americana, a subtropical tree species native to Meso- and Central America and the progenitor of cultivated avocado. The sample includes 21 wild accessions from Mexico, Costa Rica, Ecuador, and the Dominican Republic. Estimated levels of nucleotide polymorphism and linkage disequilibrium (LD) are obtained from fully resolved haplotype data from 4 nuclear loci that span 5960 nucleotide sites. Results show that, although avocado is a subtropical tree crop and a predominantly outcrossing plant, the overall level of genetic variation is not exceptionally high (nucleotide diversity at silent sites, pi(sil) = 0.0102) compared with available estimates from temperate plant species. Intralocus LD decays rapidly to half the initial value within about 1 kb. Estimates of recombination rate (based on the sequence data) show that the rate is not exceptionally high when compared with annual plants such as wild barley or maize. Interlocus LD is significant owing to substantial population structure induced by mixing of the 3 botanical races of avocado.     

Modification of matrix polysaccharides during avocado (Persea americana) fruit ripening: an assessment of the role of Cx-cellulase

The role of C_x-cellulase (EC 3.2.1.4) in fruit ripening and softening is unknown. In the present study, avocado ( Persea americana ) fruit, a rich source of C_x-cellulase, were examined to determine if the enzyme plays a role in ripening-related hemicellulose metabolism. Hemicelluloses (4 M alkali-soluble) from avocado fruit exhibited a very broad distribution of polymer sizes and an overall decrease in M_r during ripening. Polymers affected were primarily those of large M_r (relative molecular mass). The characteristic total hemicellulose M_r distribution and changes with ripening were also evident for xyloglucan (XG), a putative substrate for avocado C_x-cellulase. Hydrolytic activity toward hemicelluloses from preripe fruit was detected in crude buffer-soluble protein extracts derived from ripe avocado mesocarp tissue. XG was also degraded, and in a pattern similar to that observed during ripening. Purified C_x-cellulase also exhibited activity against specific components of isolated hemicelluloses; however, in contrast to the crude protein. C_x-cellulase alone was without influence on the M_r distribution of avocado XG. Protein depleted of C_x-cellulase was capable of moderate XG depolymerization. We conclude from the present studies that the enzyme C_x-cellulase is not involved in the ripening-related depolymerization of XG in avocado fruit.     

Enzyme activities and polyphenols related to mesocarp discolouration of avocado fruit

Avocado fruit showing severe symptoms of the mesocarp discolouration disorder exhibited significantly higher extractable activities of soluble polyphenol oxidase and peroxidase, as well as higher levels of total phenols, hydroxycinnamic acids and proanthocyanidins, when compared to healthy fruit. However, l-phenylalanine ammonia-lyase activity was very variable, and no significant differences were observed between healthy and affected fruit. Extraction of healthy, but not severely affected, fruit in the presence of 0.1 % SDS resulted in increased polyphenol oxidase activity reflecting the release of bound and/or latent enzyme. Qualitative differences between healthy and affected fruit included different patterns of polyphenol oxidase multiple forms and different polyphenol profiles. The pattern of polyphenol oxidase multiple forms from SDS-extracted healthy fruit was similar to that from mildly affected fruit not extracted with detergent.     

Cellulase occurs in multiple active forms in ripe avocado fruit mesocarp

The existence of multiple forms of avocado (Persea americana Mill. cv Hass) cellulase in crude protein extracts of ripe avocado fruit is reported. Cellulase was separated into at least 11 multiple forms by native isoelectric focusing in the pH range between 4 and 7 and visualized by both activity staining using Congo red and immunostaining. The enzyme components were acidic proteins with isoelectric points in the range of pH 5.10 to 6.80, the predominant forms having isoelectric points of 5.60, 5.80, 5.95, and 6.20. All 11 forms were immunologically related with molecular masses of 54 kilodaltons.     

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.     

Oleic acid synthesis in the fruit of Persea americana MILL

The tissue slices from the mesocarp of avocado could incorporateradioactive acetate into lipids. Oleic, palmitic and stearicacids were the most labeled fatty acids found to accumulatein triglycerides. The conclusions that oleic acid was formedby way of chain elongation of already unsaturated short-chainfatty acids, and that there was no evidence for the desaturationof stearic acid were based on the following observations: 1)Stearic acid-¹⁴C was incorporated into triglycerides by thetissue slices without formation of oleic acid. 2) The oleicacid synthesized from specifically labeled acetate was not randomlylabeled. The specific radioactivity of azelaic acid moiety ofoleic acid was rapidly increased while that of pelargonic acidmoiety was gradually increased. 3) An unexpected rise of stearicacid was observed among commonly occurring fatty acids in thetissue slices. This was accentuated by anaerobiosis which prevailedduring vacuum infiltration of labeled acetate. ¹Present address: Department of Botany, Faculty of Agriculture,Hokkaido University, Sapporo, Japan. (Received January 13, 1969; )     

Development of an efficient transient transformation protocol for avocado (Persea americana Mill.) embryogenic callus

An efficient protocol for transient transformation of avocado embryogenic callus has been established, using the PDS-1000/He system and the reporter gus gene driven by the sunflower polyubiquitin promoter. Best physical parameters for transient transformation were 900 psi helium pressure and 6 cm target distance. The level of transient gus expression was slightly higher when the amount of DNA per shot was increased from 0.6 to 1.8 μg, but it was not significantly modified by the type of microprojectile used (tungsten vs. gold particles). The transient transformation assay developed in this research was used to test the strength of different promoters and the expression of fluorescent reporter genes. Four constitutive promoters, sunflower polyubiquitin, CaMV35S, CaMV35S with enhancer, and rice actin 1, as well as a trichome-specific promoter, ATP, were analyzed. Polyubiquitin and ATP promoters yielded the highest number of gus expressing foci, while no expression was detected with the Act1 promoter from rice. Embryogenic callus was also bombarded with plasmids pXK7S*NF2 and pXK7RNR2, harboring the enhanced green fluorescent gene, EGFP, and the red fluorescent gene DsRed, respectively. Both fluorescent proteins were detected 24 and 72 h after bombardment, but the observed transformation efficiency was slightly higher in GFP bombarded cells. The transient transformation system described here can be used as a fast way to select suitable promoters and/or fluorescent genes needed to undertake stable transformation studies in avocado using currently available protocols.