Avocado Tissues Research Articles

The Enigmatic Aliphatic Acetogenins and Their Correlations With Lipids During Seed Germination and Leaf Development of Avocado (Persea americana Mill.).

Lipids in avocados have been widely studied due to their nutritional value and several reported bioactivities. Aliphatic acetogenins are a relevant component of the avocado lipidome and have been tested for several potential food and pharma industries applications. This work followed the evolution of avocado fatty acids (FAs) and aliphatic acetogenins during seed germination and leaf growth. Oil extracts of embryonic axes, cotyledons, and leaves from seedlings and trees were divided to analyze free acetylated acetogenins (AcO-acetogenins), and free FAs. Embryonic axes from germinating seeds contained the highest amount of AcO-acetogenins and FAs; this tissue also accumulated the most diverse FA profile with up to 22 detected moieties. Leaves presented the highest variations in AcO-acetogenin profiles during development, although leaves from seedlings accumulated the simplest FA profile with only 10 different FAs. Remarkably, AcO-acetogenins represented half of the carbons allocated to lipids in grown leaves, while embryonic axes and cotyledons always contained more carbons within FAs during germination. Thus, we hypothesized the use of the AcO-acetogenin acyl chain for energy production toward β-oxidation. Also, α-linolenic and docosahexaenoic acids (DHAs) were proposed as close AcO-acetogenin intermediaries based on a correlation network generated using all these data. Another part of the oil extract was fractionated into different lipid classes before transesterification to profile FAs and acetogenins bound to lipids. Acetogenin backbones were identified for the first time in triglycerides from cotyledons and mainly in polar lipids (which include phospholipids) in all developing avocado tissues analyzed. Seed tissues accumulated preferentially polar lipids during germination, while triglycerides were consumed in cotyledons. Seedling leaves contained minute amounts of triglycerides, and polar lipids increased as they developed. Results from this work suggest acetogenins might be part of the energy and signaling metabolisms, and possibly of membrane structures, underlining the yet to establish role(s) of these unusual lipids in the avocado plant physiology.

Tissue-specific proteome characterization of avocado seed during postharvest shelf life

Avocado is a nutritious and economically important fruit, generating significant income for exporter countries. Recently, by-products of this fruit such as seeds and peels, have raised interest in different industries. However, the biochemical features of the nutraceutical value of these tissues have not been analyzed using molecular approaches during the postharvest shelf life (PSL). We carried out comparative proteomics using tandem mass tagging (TMT) and synchronous-precursor selection (SPS)-MS3. We analyzed testa, cotyledon, and embryo axes from avocado seeds at detachment from the tree (unripe), and after five (breaker) and ten days (ripe) of PSL. We identified 1968 proteins, from which 933 were specific to the testa, 167 to the embryo axis, and 23 to the cotyledon. The testa had a more dynamic proteome than the other tissues, resembling similar stress responses to those observed in peel tissues, such as down-accumulation of translational machinery, cell wall catabolism and synthesis of secondary metabolites. In contrast, the up-accumulation of the biosynthesis of l-glutamine, L-isoleucine, and l-serine was observed in all tissues. Our study provides the basic biochemical and physiological features of avocado seed during PSL and demonstrates that avocado seed tissues could potentially be used as a costless source of high-value compounds. SignificanceAvocado seed as a fruit by-product is a source of different valuable molecules, including those with nutraceutical properties. During PSL, several biochemical and physiological modifications occur in this dispersal unit, which also includes the alteration of several key metabolites' content. However, the proteome profile associated with different metabolic pathways that regulate the inner content of seed metabolites has not been previously studied. Our tissue-specific proteomics TMT-SPS-MS3-based provides the first evidence of molecular and physiological changes in avocado tissues during PSL delivering fundamental knowledge of this organ. In this vein, the modulation of secondary metabolites, amino acid, and sugar metabolism of avocado tissues during PLS can encourage these by-products exploitation in multiple industries.

Chemical Profile and Safety Assessment of a Food-Grade Acetogenin-Enriched Antimicrobial Extract from Avocado Seed.

Acetogenins are bioactive fatty acid derivatives found in avocado tissues. Their efficacy as antimicrobials has been documented and initiated interest to use them as replacements of synthetic food additives. The present work focused on evaluation of multiple analytical methodologies for detection and quantification of organic solids present in a food-grade acetogenin-enriched extract (Avosafe®), and on its safety evaluations using bacterial reverse mutation (AMES) tests and acute oral toxicity to rat assays. Results confirmed chemical structures of two acetogenins as present in Avosafe® (AcO-avocadyne-(0) and AcO-avocadiene B-(3)), and together with seven other previously known compounds, quantified 94.74 ± 5.77% w/w of its solids as acetogenins. Safety evaluations indicated that Avosafe® was non-mutagenic and had an acute median lethal oral dose (LD50) to rats higher than the maximum concentration tested (>2000 mg·kg−1), with no signs of macroscopic abnormalities in organs. Mean body weight and hematological and biochemical parameters were normal after 14 days of a single oral dose of 2000 mg·kg−1. The results advance scientific information on the safety of avocado seed acetogenins and also generate new knowledge on profiles and concentrations of individual acetogenins found in avocado tissues (seed, pulp, and leaves) and in Avosafe®.

A phenol/chloroform-free method to extract nucleic acids from recalcitrant, woody tropical species for gene expression and sequencing

BackgroundWoody tropical plants contain high levels of complex organic compounds that inhibit the chemical procedures needed to extract RNA or DNA, thus compromising downstream applications such as RNA sequencing and analysis of gene expression. To overcome this issue, researchers must use extraction protocols using CTAB/PVP buffer instead of commercially available DNA/RNA extraction kits. However, these protocols are time-consuming, use toxic chemicals like phenol and chloroform, and can only be used to process a small number of samples at a time. To overcome these issues, we developed a new CTAB/PVP based protocol for RNA or DNA extraction that eliminates the traditional phenol/chloroform step. Furthermore, the protocol was developed for 96-well plates to speed up processing.ResultsOur new protocol enabled us to successfully extract RNA from macadamia, avocado, and mango tissues that are traditionally difficult to work with. This RNA was then successfully used to synthesise cDNA for real-time quantitative PCR and to generate good quality RNA-Seq libraries. Our protocol can be easily converted for rapid DNA extraction from different tropical and sub-tropical tree species.ConclusionThis method enables safer and faster DNA and RNA extraction from recalcitrant species, thus facilitating future work on tropical trees.

Changes in sugars, total protein, and oil in ‘Hass’ avocado (Persea americana Mill.) fruit during ripening

SummaryA better understanding of the post-harvest physiology of avocado (Persea americana Mill.) fruit is required to improve the post-harvest management of the crop. Two heptoses, D-mannoheptulose and perseitol, occur in unusually high concentrations in avocado tissues and are thought to be important in fruit ripening. We determined changes in the concentrations and relationships between individual sugars, total soluble proteins, and oil during fruit ripening. ‘Hass’ fruit were harvested from cool and warm sub-tropical areas in South Africa three-times during a single season. Dry matter increased during the season at both locations. For fruit from the cool sub-tropical climate, D-mannoheptulose and perseitol concentrations declined rapidly from 15.9 mg g–1 dry weight (DW) and 7.9 mg g–1 DW to 2.1 mg g–1 DW and 0.6 mg g–1 DW, respectively, 13 d after harvest. Overall concentrations of glucose and fructose were negligible at harvest and increased to 1.4 mg g–1 DW and 0.8 mg g–1 DW, respectively, during ripening. The concentration of glucose in fast-ripening fruit increased 3 d prior to increasing in slow-ripening fruit. The concentration of sucrose was intermediate (2.8 – 4.5 mg g–1 DW) and was variable during ripening. The concentration of total soluble proteins increased during ripening from 17.8 mg g–1 DW to 34.8 mg g–1 DW, whereas the concentration of oil was fairly stable during ripening, and varied from 24.8 – 36.7% DW, depending on fruit maturity. It is suggested that the heptose pool is an important source of carbon and/or energy in avocado fruit. The low concentrations of these sugars and the rapid decline in their levels, post-harvest, indicate that optimal post-harvest management, especially cold storage and ripening temperature regimes, is necessary to reduce the risk of post-harvest disorders and to ensure a consistent supply of high-quality avocado fruit.

TEXTURAL, BIOCHEMICAL AND MICRO-STRUCTURAL CHANGES IN MESOCARP TISSUE OF IMPORTED AVOCADO FROM PERU DURING RIPENING

Avocado (Persea americana Mill.) fruit is a valuable product and notorious for the difficulties encountered in determining quality. Typically the degree of ripeness of many climacteric fruits, such as avocado, is measured by assessing flesh firmness. The aim of the presented work was to elucidate the temporal and spatial changes in texture, biochemistry and micro-structure in different avocado tissues from the same fruit. Fruit were first treated with ethylene and then ripened at 12°C. Samples were taken four times over 10 days storage. Maximum load and viscoelasticity of horizontally-cut slices from fruit (n=24) imported from Peru were measured during ripening. These texture parameters were measured using an Instron 5542 universal testing machine fitted with a 500 N or 5 N load cell. Non-structural carbohydrates (NSCs; viz. sucrose, mannoheptulose, perseitol) and fatty acid methyl esters from the same samples were identified and quantified using standard HPLC coupled to evaporative light scattering detection and gas chromatography coupled to flame ionisation detection, respectively. Samples taken from adjacent mesocarp tissue slices were examined: each specimen included the sclerenchymatic exocarp, fleshy mesocarp and endocarp including the coat of the seed. An analysis of variance was performed to elucidate the change of parameters during fruit ripening. NSC content and textural properties changed during ripening and showed spatial heterogeneity within individual fruit. Micro-structural changes were evident during the latter stages of ripening and coincided with fruit softening and degradation in mannoheptulose. These findings might be used to enhance quality monitoring of imported avocado fruit.

First Report of Xylella fastidiosa in Avocado in Costa Rica.

Since the late 1990s, chlorotic mottling, marginal scorch, deformation of leaves, defoliation, shortening of internodes, and branch dieback have been observed in avocado trees (Persea americana Mill.) in Costa Rica. The symptoms are not uniformly distributed in the tree, so some branches are symptomatic while others are not. These symptoms are similar to several leaf scorch diseases caused by the bacterium Xylella fastidiosa Wells (2,4). This bacterium has been detected in coffee and citrus plants in Costa Rica. Of 227 avocado trees tested by double-antibody sandwich (DAS)-ELISA with X. fastidiosa specific antiserum (Agdia Inc., Elkhart, IN) from 2000-2004, 188 were positive. Results of ELISA tests of individual trees varied with the season and branches tested. Fifteen greenhouse-grown, ELISA-negative avocado seedlings were grafted with budwood from an ELISA-positive tree. Eight of these developed scorch symptoms and one also showed chlorotic mottling and deformation, showing that the disease is graft transmitted. All of these features are characteristic of diseases caused by X. fastidiosa (2,4). Transmission electron microscopy of leaf petioles from three field trees positive by ELISA, revealed rod-shaped bacilli approximately 1.6 to 2.0 μm long and 0.3 μm in diameter with a rippled cell wall inside xylem vessels and embedded in a matrix; morphology and measurements that are consistent with those reported for X. fastidiosa (2). DNA extraction and PCR attempts have been limited by mucilaginous sap from avocado. Positive PCR results (approximately 472-bp band) were obtained from two of the grafted seedlings and seven field trees from two distinct geographical locations (Alajuela and San José provinces) with DNA extractions from the plant sap using DNeasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany) following a modified protocol (1) and nested PCR (3). Four of the PCR products, including one from the grafted seedlings, were cloned and sequenced in duplicate. GenBank sequences EU021997 to EU022000 present 99 to 100% sequence identity to a Pierce's disease strain from California (Temecula1) and 94 to 95% to a citrus variegated chlorosis strain from Brazil (Found-5). Several attempts have been made to isolate the bacterium in 'periwinkle wilt' and buffered cysteine-yeast extract media with negative results, probably because of the rapid production of mucilaginous sap when the avocado tissues were sampled. To our knowledge, this is the first report of X. fastidiosa in avocado trees.

Effects of low oxygen on in vitro translation products of poly(A) + RNA, cellulase and alcohol dehydrogenase expression in preclimacteric and ripening-initiated avocado fruit

In order to distinguish the interactions between low oxygen and fruit developmental stages, preclimacteric and ripening-initiated with propylene avocado fruit were exposed to different low oxygen levels and the steady-state levels of protein and mRNA accumulation of selected hypoxic and ripening genes were investigated. In addition, the patterns of translatable mRNA were studied by 2D IEF/SDS-PAGE analysis in preclimacteric fruit. Analysis of mRNA populations in preclimacteric avocado fruit revealed that low oxygen levels induced new mRNA species possibly implicated in the adaptive mechanism under low oxygen, suppressed de novo synthesized ones, or left unaffected house-keeping and/or pre-existing mRNAs, indicating that the low oxygen response is complex and involves more than a simple adaptation in energy metabolism. The accumulation of cellulase protein and mRNA was irrespective of low oxygen concentrations in preclimacteric fruit and oxygen dependent in ripening-initiated fruit. That is, preclimacteric avocado fruit contained cellulase protein and mRNA which were not suppressed by low oxygen treatment. Low oxygen tensions prevented the accumulation of cellulase protein and mRNA in ripening-initiated with propylene avocado tissues. New ADH isoenzymes were present in preclimacteric and ripening-initiated avocado fruit held in low oxygen atmospheres and correlated with elevated ADH mRNA levels. Low oxygen treatment (0–5%) did not result in increased ADH activity in avocado fruit. Neither the duration of exposure nor the developmental stage of the fruit altered this pattern, suggesting that unknown factor(s) might accumulate which possibly interfere with ADH activity under such conditions.

Application of RT-PCR for Indexing Avocado Sunblotch Viroid.

A method for the routine detection of avocado sunblotch viroid (ASBVd) in nucleic acid extracts of infected avocado tissues by reverse transcription-polymerase chain reaction (RT-PCR) was developed using ASBVd-specific primers. Amplified cDNA products were analyzed by electrophoresis on nondenaturing 6% polyacrylamide slab gels. The size of the major RT-PCR product from ASBVd-infected tissue was estimated to be 250 bp. This product was absent from amplified extracts of uninfected tissue. The amplification product from ASBVd was sequenced by the dideoxynucleotide chain termination method, and the sequence was over 97% identical to the published sequence. The RT-PCR assay is sensitive enough to allow viroid detection without requiring large amounts of tissue, highly purified ASBVd, or molecular hybridization.

Avocado sunblotch disease: a persistent viroid infection in which variants are associated with differential symptoms.

Variants of avocado sunblotch viroid (ASBVd) of between 247 and 250 nucleotides in length have been recovered from diseased avocado tissues. The sunblotch syndrome covers a complex pattern of disease symptoms which are associated with infection by variants of ASBVd. The viroid species are designated ASBVd-B, ASBVd-V and ASBVd-Sc from their association with bleached, variegated or symptomless carrier tissues respectively. Host-viroid interactions and structural relationships among the variants suggest a transition in sunblotch disease from a severe acute to a persistent mild form of infection.

The absolute configuration effect on the activity of the avocado rooting promoter

Abstract 16-Heptadecyn-1,2,4-triol is the most active component of the avocado rooting promoter (ARP). All four diastereoisomers of this compound have been synthesized. Their root promoting activity was determined over the physiologically active concentration range. It was found that the (2 R ,4 R )-stereoisomer exerts a rooting activity similar to that of the extracted and purified compound from avocado tissues. The (2 R ,4 S ), and (2 S ,4 R )-stereoisomers had lower activity and the (2 S ,4 S )-stereoisomer had the lowest activity. It is concluded that the natural form, (2 R ,4 R ), acts in the rooting process either in its original structure or after reaction which does not alter its chiral centres.

Interference of Phenolic Compounds with the 1-Aminocyclopropane-1-Carboxylic Acid Assay

The yields of ethylene from endogenous and exogenous 1-aminocyclo-propane-1-carboxylic acid (ACC) in avocado (Persea Americana Mill.) fruit pedicel extracts were very low when assayed by the method of Lizada and Yang (1979 Anal Biochem 100: 140-145). Addition of phenolic compounds, which are present in avocado tissues, to the assay mixture significantly reduced the conversion efficiency of ACC to ethylene. A negative correlation was found between the amount of the plant material in the assay mixture and the conversion efficiency of ACC to ethylene. Removal of phenolic compounds from pedicel extracts by polyvinylpolypyrrolidone, Amberlite XAD-7, and Dowex-50 column chromatography or lead acetate precipitation greatly increased the yields of thylene from ACC in these extracts. The use of polyvinylpolypyrrolidone column chromatography also enabled us to obtain more accurate estimations of endogenous ACC levels in carnation (Dianthus caryophyllus L.) petal extracts. The conversion efficiency of ACC to ethylene could be improved by increasing the concentrations of mercuric chloride and NaOCl in the assay mixture.

The chemical identification of root promoters extracted from avocado tissues

From the Avocado Rooting Promoter (ARP) 4 compounds were isolated and identified as: The rooting activity of the pure compounds was verified using the mung bean rooting bioassay. Compound 2II is the most active.

Inhibition of Ethylene Production in Fruit Slices by a Rhizobitoxine Analog and Free Radical Scavengers

The rhizobitoxine analog, L-2-amino-4-(2-aminoethoxy)-trans-3-butenoic acid (Ro), which effectively inhibits ethylene production in apple (Malus domestica Borkh.) and other tissues at concentrations at about 68 micromolar, inhibited ethylene production by about 50 to 70% in green tomato (Lycopersicon esculentum Mill.) fruit slices but only by about 15% in pink and ripe tomato tissue slices. Ethylene production in climacteric-rise and postclimacteric avocado slices was likewise relatively insensitive to 68 micromolar Ro. At 340 micromolar Ro, inhibition of ethylene production increased up to 50% in pink tomato slices, whereas 680 micromolar Ro was required to inhibit ethylene production by 30% in avocado slices. Incorporation of (14)C from [(14)C]methionine into ethylene in green and pink tomato tissues was inhibited by Ro to about the same extent as inhibition of total ethylene production. Results thus far are inconclusive as to the mechanism of Ro resistance in tomato and avocado tissues. At 1 millimolar, free radical scavengers such as benzoate, propyl gallate, nordihydroguaiaretic acid, and to a lesser extent, eugenol, inhibited ethylene production in both Ro-sensitive (green tomato and apple) tissues and Ro-resistant (pink tomato and avocado) tissues. Therefore, free radical steps are suggested in the ethylene-forming systems.

Ethylene Biosynthesis in Fruit Tissues

Tracer studies with avocado tissues indicate that methionine is converted to ethylene at stages of the climacteric rise and the climacteric peak, but not at the preclimacteric stage. The results suggest that the control of ethylene biosynthesis is at a step after methionine is synthesized. The endogenous content of methionine was found to be so low that methionine must be actively turned over for ethylene biosynthesis during the stages when the rate of ethylene production is high. Oxygen was found to be essential for this conversion, indicating that at least one of the steps in conversion of methionine to ethylene is oxygen-dependent. The ability of methionine and its keto analogue (alpha-keto-gamma-methylthiobutyric acid) to serve as ethylene precursors by apple tissues was compared. Chemical and kinetic evidence support the view that methionine is a closer precursor of ethylene than its keto analogue.

Recovery of Ethylene Chlorobromide from Agricultural Products

A quantitative method has been developed to determine residual ethylene chlorobromide in agricultural and food commodities that have been fumigated for insect infestations. The ethylene chlorobromide was separated from the commodities by steam distillation, hydrolyzed in alcoholic alkali and determined as bromine by iodometry. When ethylene chlorobromide, varying in amounts between 0.2 mg and 5.35 mg, was added to water and to macerated orange, lemon, and avocado tissues and ground corn and wheat, the recoveries varied from 93.9% in corn to 100.3% in water. Experiments performed with the macerated tissues, to which known amounts of ethylene chlorobromide were added and allowed to stand at approximately 70°F for 10 days, indicated that the fumigant reacted with the chemical constituents of these commodities. Under experimental conditions wheat retained 2.7 ppm ethylene chlorobromide and 0.8 ppm bromine as a degradation product 14 days after fumigation, and Valencia oranges retained 1.0 ppm ethylene ehlorohromide in the whole fruit and 11.2 ppm bromine in the peel 9 days after fumigation.