Ontogenic synchronization of Bephratelloides cubensis, Annona macroprophyllata seeds and acetogenins from Annonaceae.

The seeds of Annona macroprophyllata Donn. Sm. contain idioblasts with toxic acetogenins, including laherradurin and rolliniastatin-2, in relatively high proportions. Both metabolites are cataloged as potent insecticides for several species, even so, the wasp Bephratelloides cubensis Ashmead fulfills almost its entire life cycle inside the seeds of this and other annonaceous species, to such a degree, that they constitute a strong selection pressure. In order to document the chemical relationship between the two species, it is reported for the first time in this paper the presence of idioblasts and acetogenins during the ontogenic development of the seeds of A. macroprophyllata, and contrasted with the development of B. cubensis. The results indicate that idioblasts with laherradurin and rolliniastatin-2 acetogenins are formed in the middle stages of the endospermic development, also that both acetogenins are biosynthesized simultaneously, and that their proportion is dependent on the degree of development. The acetogenins are present in high amounts that suppose a sufficient toxic barrier and, in this case, laherradurin is the most abundant (> 1000 µg g dry weight-1). The wasp B. cubensis only emerges from the seeds to copulate and returns for oviposition; its larval phase coincides with the appearance of acetogenins, so it feeds on the acetogenic endosperm. The absence of acetogenins in the tissues and excreta of the insect supposes a metabolization of the molecules, which would explain the tolerance to its toxicity.

The role of the integuments in pollen tube guidance in flowering plants.

In angiosperms, pollen tube entry into the ovule generally takes place through the micropyle, but the exact role of the micropyle in pollen tube guidance remains unclear. A limited number of studies have examined eudicots with bitegmic micropyles, but information is lacking in ovules of basal/early-divergent angiosperms with unitegmic micropyles. We have evaluated the role of the micropyle in pollen tube guidance in an early-divergent angiosperm (Annona cherimola) and the evolutionarily derived Arabidopsis thaliana by studying γ-aminobutyric acid (GABA) and arabinogalactan proteins (AGPs) in wild-type plants and integument-defective mutants. A conserved inhibitory role of GABA in pollen tube growth was shown in A. cherimola, in which AGPs surround the egg apparatus. In Arabidopsis, the micropyle formed only by the outer integument in wuschel-7 mutants caused a partial defect in pollen tube guidance. Moreover, pollen tubes were not observed in the micropyle of an inner no outer (ino) mutant in Arabidopsis, but were observed in homologous ino mutants in Annona. The similar distribution of GABA and AGPs observed in the micropyle of Arabidopsis and Annona, together with the anomalies from specific integument mutants, support the role of the inner integument in preventing multiple tube entrance (polytubey) in these two phylogenetically distant genera.

Beetle pollination and flowering rhythm of Annona coriacea Mart. (Annonaceae) in Brazilian cerrado: Behavioral features of its principal pollinators.

The conservation and sustainable management of Annona coriacea requires knowledge of its floral and reproductive biology, and of its main pollinators and their life cycles. In this work, we analyzed these aspects in detail. Floral biology was assessed by observing flowers from the beginning of anthesis to senescence. The visiting hours and behavior of floral visitors in the floral chamber were recorded, as were the sites of oviposition. Excavations were undertaken around specimens of A. coriacea to determine the location of immature pollinators. Anthesis was nocturnal, starting at sunset, and lasted for 52-56 h. The flowers were bisexual, protogynous and emitted a strong scent similar to the plant´s own ripe fruit. There was pronounced synchrony among all floral events (the period and duration of stigmatic receptivity, release of odor, pollen release and drooping flowers) in different individuals, but no synchrony in the same individuals. All of the flowers monitored were visited by beetle species of the genera Cyclocephala and Arriguttia. Beetles arrived at the flowers with their bodies covered in pollen and these pollen grains were transferred to the stigmata while foraging on nutritious tissues at the base of the petals. With dehiscence of the stamens and retention within the floral chamber, the bodies of the floral visitors were again covered with pollen which they carried to newly opened flowers, thus promoting the cycle of pollination. After leaving the flowers, female beetles often excavated holes in the soil to lay eggs. Larvae were found between the leaf litter and the first layer of soil under specimens of A. coriacea. Cyclocephala beetles were the main pollinators of A. coriacea, but Arriguttia brevissima was also considered a pollinator and is the first species of this genus to be observed in Annonaceae flowers. Annona coriacea was found to be self-compatible with a low reproductive efficiency in the area studied. The results of this investigation provide ecological data that should contribute to the conservation and economic exploitation of A. coriacea.

The Unusual Acid-Accumulating Behavior during Ripening of Cherimoya (Annona cherimola Mill.) is Linked to Changes in Transcription and Enzyme Activity Related to Citric and Malic Acid Metabolism.

Cherimoya (Annona cherimola Mill.) is a subtropical fruit characterized by a significant increase in organic acid levels during ripening, making it an interesting model for studying the relationship between acidity and fruit flavor. In this work, we focused on understanding the balance between the concentration of organic acids and the gene expression and activity of enzymes involved in the synthesis and degradation of these metabolites during the development and ripening of cherimoya cv. "Concha Lisa". Our results showed an early accumulation of citric acid and other changes associated with the accumulation of transcripts encoding citrate catabolism enzymes. During ripening, a 2-fold increase in malic acid and a 6-fold increase in citric acid were detected. By comparing the contents of these compounds with gene expression and enzymatic activity levels, we determined that cytoplasmic NAD-dependent malate dehydrogenase (cyNAD-MDH) and mitochondrial citrate synthase (mCS) play important regulatory roles in the malic and citric acid biosynthetic pathways.

Attraction of Pollinators to Atemoya (Annona squamosa × Annona cherimola) in Puerto Rico Using Commercial Lures and Food Attractants.

Atemoya is a hybrid between Annona squamosa L. and Annona cherimola Miller (Annonaceae) and has potential to be an important fruit crop in tropical and subtropical areas. A major impediment to fruit production is low fruit set due to inadequate pollinator visits, typically, by beetles in the family Nitidulidae. We used Universal moth traps to monitor the attractiveness of two commercially available Nitidulidae lures in combination with various food attractants, including raw bread dough, apple juice, and malta beverage, a soft drink by-product of the brewing process. The most commonly trapped beetles were, in order of decreasing frequency, Carpophilus dimidiatus (F.), Brachypeplus mutilatus Erichson, Urophorus humeralis (F.) (Coleoptera: Nitidulidae), and Europs fervidus Blatchley (Coleoptera: Monotomidae). All traps, except the unbaited control traps, caught beetles. In a previous study, we found that combining two commercial lures had a synergistic effect on the attraction of these beetle species. In this study, the addition of food attractants increased the number of beetles trapped compared with traps baited with only the commercial lures. Also, food attractants appear to be key in attracting U. humeralis; only one U. humeralis individual of the 206 caught during the experiment was trapped without a food attractant. The variation between the number of beetles caught in traps containing the same treatments was high and may explain the erratic results reported in other studies of pollination in Annona spp. The results are discussed with respect to the use of nitidulid lures and food attractants to increase fruit set in atemoya and other Annonaceae.

Effect of storage in overcoming seed dormancy of Annona coriacea Mart. seeds.

The aim of this study was to evaluate the effect of pre-treatments on overcoming dormancy of A. coriacea seeds. Seeds were processed and stored in polyethylene bags at temperatures of at -18°C (42% RH), 5°C (34% RH), 15°C (60% RH) and 25°C (34% RH), during 0, 30, 60, 90, 120 and 150 days. After storage, seeds were immersed in 350 mg.L-1 gibberellic acid for 144 hours. Sowing was carried out in plastic bags containing Red Latosol + Bioplant®. Moisture content, emergence percentage, emergence speed index, length and dry mass of seedlings, were evaluated. The experimental design was completely randomized in a factorial with four replications of 50 seeds each. The seed storage at 5°C and subsequent immersion in gibberellic acid was efficient to reach high percentage, emergence speed and plant growth. A. coriacea seeds showed non-deep simple morphophysiological dormancy wherein the physiological component can be overcome after the seeds are storage at 5°C for a maximum period of 53 days and subsequent immersed in exogenous GA (350 mg.L-1 for 144 hours).

Influence of seasonal variation on the phenology and liriodenine content of Annona lutescens (Annonaceae).

Annona lutescens Saff. (Annonaceae) grows as a native tree in Chiapas, Mexico in Tropical Dry Forest habitat. Like most Annonaceae, it biosynthesizes benzylisoquinoline alkaloids, mostly liriodenine. To determine the influence of seasonal changes in the accumulation of liriodenine, the monthly variation of liriodenine content in roots, stems and leaves of mature and young trees was observed. These parts of young and mature A. lutescens trees were collected monthly over a 1 year period and the alkaloids were extracted; the liriodenine was quantified by high-resolution liquid chromatography. The phenological stages of the species were also assessed (leaf development, flowering and fruiting) using the Biologische Bundesanstalt, Bundessortenamt und Chemische Industrie (BBCH) scale. The analysis of both young and mature trees showed a significant increase in the liriodenine concentration occurs within roots during the dry season, which coincides with leaf fall. A significant decrease also occurred at the beginning of the rainy season (the period of leaf growth); the liriodenine content for the next rainy season did not reach the levels of the previous dry season. The climatic variation induced phenological and physiological changes in this species.

Pollen performance, cell number, and physiological state in the early-divergent angiosperm Annona cherimola Mill. (Annonaceae) are related to environmental conditions during the final stages of pollen development.

Pollen performance is an important determinant for fertilization success, but high variability in pollen behavior both between and within species occurs in different years and under varying environmental conditions. Annona cherimola, an early-divergent angiosperm, is a species that releases a variable ratio of bicellular and tricellular hydrated pollen at anther dehiscence depending on temperature. The presence of both bi- and tricellular types of pollen is an uncommon characteristic in angiosperms and makes Annona cherimola an interesting model to study the effect of varying environmental conditions on subsequent pollen performance during the final stages of pollen development. In this work, we study the influence of changes in temperature and humidity during the final stages of pollen development on subsequent pollen performance, evaluating pollen germination, presence of carbohydrates, number of nuclei, and water content. At 25 °C, which is the average field temperature during the flowering period of this species, pollen had a viability of 60-70 %, starch hydrolyzed just prior to shedding, and pollen mitosis II was taking place, resulting in a mixture of bi- and tricellular pollen. This activity may be related to the pollen retaining 70 % water content at shedding. Temperatures above 30 °C resulted in a decrease in pollen germination, whereas lower temperatures did not have a clear influence on pollen germination, although they did have a clear effect on starch hydrolysis. On the other hand, slightly higher dehydration accelerated mitosis II, whereas strong dehydration arrested starch hydrolysis and reduced pollen germination. These results show a significant influence of environmental conditions on myriad pollen characteristics during the final stages of pollen development modifying subsequent pollen behavior and contributing to our understanding of the variability observed in pollen tube performance.

Seedless fruits and the disruption of a conserved genetic pathway in angiosperm ovule development.

Although the biological function of fruiting is the production and dissemination of seeds, humans have developed seedless fruits in a number of plant species to facilitate consumption. Here we describe a unique spontaneous seedless mutant (Thai seedless; Ts) of Annona squamosa (sugar apple), a member of the early-divergent magnoliid angiosperm clade. Ovules (seed precursors) of the mutant lack the outer of two normal integuments, a phenocopy of the inner no outer (ino) mutant of Arabidopsis thaliana. Cloning of the INO ortholog from A. squamosa confirmed conservation of the outer integument-specific expression pattern of this gene between the two species. All regions of the gene were detectable in wild-type A. squamosa and in other members of this genus. However, no region of the INO gene could be detected in Ts plants, indicating apparent deletion of the INO locus. These results provide a case of a candidate gene approach revealing the apparent molecular basis of a useful agronomic trait (seedless fruit) in a crop species, and indicate conservation of the role of a critical regulator of ovule development between eudicots and more ancient lineages of angiosperms. The outer integument is one synapomorphy of angiosperms separating them from other extant seed plants, and the results suggest that the evolution of this structure was contemporaneous with the derivation of INO from ancestral YABBY genes. Thus, a unique lateral structure appears to have coevolved with a novel gene family member essential for the structure's formation.

[Chemical constituents of an endophytic fungus from Annona muricata].

OBJECTIVE: To investigate the chemical constituents of an endophytic fungus, F-31, from Annona muricata and search antitumor natural products. METHOD: After scaling up, the fermentation broth and mycelia were extracted by macroporous resin and chromatographied by silica gel column, Sephadex LH-20 gel column and semi-preparative HPLC. The structures of compounds were determined by the means of extensive spectroscopic data The activity of the compounds were evaluated through MTT assay. RESULT: Six compounds were isolated from the fermentation broth and mycelia of this fungus, their structures were identified as 5-(3-hydroxybutyl)furan-2(5H)-one(1), chloranthalactone E(2), 5, 7-dimethyl-6-hydroxycoumarin(3), 1, 2, 4-triazole-(1'R, 2'R, 3'R, 4'R)-nucleosides(4), L-tryptophan(5), L-phenylalanine(6). The in vitro pharmalogical evaluation results displayed that the above compounds exhibited no inhibitory effects on the proliferation of six tumor cell lines (HCT-8, Bel-7402, BGC-823, A549, A2780 and MCF-7). CONCLUSION: Among these obtained compounds, compound 1 was a new compound.

The progamic phase of an early-divergent angiosperm, Annona cherimola (Annonaceae).

BACKGROUND AND AIMS: Recent studies of reproductive biology in ancient angiosperm lineages are beginning to shed light on the early evolution of flowering plants, but comparative studies are restricted by fragmented and meagre species representation in these angiosperm clades. In the present study, the progamic phase, from pollination to fertilization, is characterized in Annona cherimola, which is a member of the Annonaceae, the largest extant family among early-divergent angiosperms. Beside interest due to its phylogenetic position, this species is also an ancient crop with a clear niche for expansion in subtropical climates. METHODS: The kinetics of the reproductive process was established following controlled pollinations and sequential fixation. Gynoecium anatomy, pollen tube pathway, embryo sac and early post-fertilization events were characterized histochemically. KEY RESULTS: A plesiomorphic gynoecium with a semi-open carpel shows a continuous secretory papillar surface along the carpel margins, which run from the stigma down to the obturator in the ovary. The pollen grains germinate in the stigma and compete in the stigma-style interface to reach the narrow secretory area that lines the margins of the semi-open stylar canal and is able to host just one to three pollen tubes. The embryo sac has eight nuclei and is well provisioned with large starch grains that are used during early cellular endosperm development. CONCLUSIONS: A plesiomorphic simple gynoecium hosts a simple pollen-pistil interaction, based on a support-control system of pollen tube growth. Support is provided through basipetal secretory activity in the cells that line the pollen tube pathway. Spatial constraints, favouring pollen tube competition, are mediated by a dramatic reduction in the secretory surface available for pollen tube growth at the stigma-style interface. This extramural pollen tube competition contrasts with the intrastylar competition predominant in more recently derived lineages of angiosperms.

Germination ecophysiology of Annona crassiflora seeds.

BACKGROUND AND AIMS: Little is known about environmental factors that break morphophysiological dormancy in seeds of the Annonaceae and the mechanisms involved. The aim of this study was to characterize the morphological and physiological components of dormancy of Annona crassiflora, a tree species native to the Cerrado of Brazil, in an ecophysiological context. METHODS: Morphological and biochemical characteristics of both embryo and endosperm were monitored during dormancy break and germination at field conditions. Seeds were buried in the field and exhumed monthly for 2 years. Germination, embryo length and endosperm digestion, with endo-beta-mannanase activity as a marker, were measured in exhumed seeds, and scanning electron microscopy was used to detect cell division. The effect of constant low and high temperatures and exogenous gibberellins on dormancy break and germination was also tested under laboratory conditions. KEY RESULTS: After burial in April, A. crassiflora seeds lost their physiological dormancy in the winter months with lowest monthly average minimum temperatures (May-August) prior to the first rainfall of the wet season. The loss of physiological dormancy enabled initiation of embryo growth within the seed during the first 2 months of the rainy season (September-October), resulting in a germination peak in November. Embryo growth occurred mainly through cell expansion but some dividing cells were also observed. Endosperm digestion started at the micropylar side around the embryo and diffused to the rest of the endosperm. Exogenous gibberellins induced both embryo growth and endo-beta-mannanase activity in dormant seeds. CONCLUSIONS: The physiological dormancy component is broken by low temperature and/or temperature fluctuations preceding the rainy season. Subsequent embryo growth and digestion of the endosperm are both likely to be controlled by gibberellins synthesized during the breaking of physiological dormancy. Radicle protrusion thus occurred at the beginning of the rainy season, thereby maximizing the opportunity for seedlings to emerge and establish.

Rain forest provides pollinating beetles for atemoya crops.

Small beetles, usually species of Nitidulidae, are the natural pollinators of atemoya (Annona squamosa L. x A. cherimola Mill. hybrids; custard apple) flowers but commercial atemoya growers often need to carry out labor-intensive hand pollination to produce enough high-quality fruit. Because Australian rain forest has plant species in the same family as atemoya (Annonaceae) and because many rain forest plants are beetle pollinated, we set out to discover whether tropical rain forest in far north Queensland harbors beetles that could provide this ecosystem service for atemoya crops. Orchards were chosen along a gradient of increasing distance from tropical rain forest (0.1-24 km). We sampled 100 flowers from each of nine atemoya orchards and determined the identity and abundance of insects within each flower. To assess the amount of pollination due to insects, we bagged six flowers per tree and left another six flowers per tree accessible to insects on 10 trees at an orchard near rain forest. Results indicated that atemoya orchards < or = 0.5 km from rain forest were predominantly visited by five previously unrecognized native beetle pollinators that are likely to originate in tropical rain forest. These native beetles occurred reliably enough in crops near rain forest to have a positive effect on the quantity of fruit produced but their contribution was not great enough to satisfy commercial production needs. Management changes, aimed at increasing native beetle abundance in crops, are required before these beetles could eliminate the need for growers to hand pollinate atemoya flowers. Appreciation of the value of this resource is necessary if we are to develop landscapes that both conserve native biodiversity and support agricultural production.

Flooding, root temperature, physiology and growth of two Annona species.

The effects of root zone temperature (RZT) and flooding on physiology and growth of Annona glabra L. (pond apple) and A. muricata L. (soursop) were investigated. Trees planted in containers were exposed to RZTs of 5, 10, 20, 25 or 35 degrees C in controlled root temperature chambers. Trees at each RZT were either non-flooded (control) or continuously flooded. There were four replications over time for each treatment combination. Pond apple was more flood-tolerant than soursop. A combination of flooding and RZTs of 5 and 10 degrees C resulted in tree mortality of both species by Week 4. Only trees that appeared to develop morphological adaptations survived continuous flooding. In both species, net CO2 assimilation (A) decreased to nearly zero within 1 week following exposure to RZTs of 5 or 10 degrees C and became consistently negative over the remaining experimental period. Flooding reduced leaf chlorophyll index (measured with a SPAD meter), A and plant growth, and increased root electrolyte leakage from soursop. Optimum growth occurred at RZTs of 25 to 35 degrees C for non-flooded pond apple trees and at 20 to 25 degrees C for flooded trees. Soursop exhibited maximum growth at RZTs of 35 degrees C under non-flooded conditions and at 25 degrees C under flooded conditions.

High CO2 atmosphere modulating the phenolic response associated with cell adhesion and hardening of Annona cherimola fruit stored at chilling temperature.

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5.) activity, tanning ability, and polyphenols levels were measured in cherimoya (Annona cherimola Mill.) fruit treated with 20% CO(2) + 20% O(2) + 60% N(2) for 1, 3, or 6 days during chilling temperature (6 degrees C) storage. The residual effect of CO(2) after transfer to air was also studied. These observations were correlated with texture and cellular characteristics, visualized by cryo-SEM. Tanning ability and the early increase in tannin polyphenols induced by chilling temperature were reduced by CO(2) treatment. Conversely, high CO(2) atmosphere enhanced the nontannin polyphenol fraction as compared with fruit stored in air. Lignin accumulation and PAL activation observed in untreated fruit after prolonged storage at chilling temperature were prevented by high CO(2). Moreover, the restraining effect on lignification was less effective when the CO(2) treatment was prolonged for 6 days. In addition, fruits held at these conditions had greater firmness and the histological characterization of the separation between cells was similar to that in untreated fruits. We conclude that CO(2) treatment modulates the phenolic response that seems to regulate the strength of cell adhesion and so to prevent hardening caused by chilling temperature storage.