Release mechanisms of bioactive compounds in fruits submitted to high hydrostatic pressure: A dynamic microstructural analysis based on prickly pear cells.

High hydrostatic pressure (HHP) promotes the release of bioactive compounds from their intracellular compartments making them more bioaccessible. Our aim was to propose a schematic tissue model to explain the release mechanisms of betalains and phenolic compounds in vegetable cells submitted to HHP by analyzing cell microstructure, cell morphology, cell viability and the localization of bioactive compounds in prickly pear fruits. Prickly pear slices were pressurized at 100, 350 and 600 MPa at 20 °C. Chlorenchyma cells (in peels) and parenchyma cells (in pulps) were analyzed by transmission electron microscopy, confocal laser scanning microscopy and optical microscopy. After pressurization, the respiration and ethylene production of processed fruits were measured every 6 h (during storage at 16 °C and 75% RH for 24 h). In chlorenchyma cells, HHP ruptured betalain-storing vesicles in the cytoplasm and possibly increased the activity of endogenous enzymes. Contrarily, HHP released betalains from the vacuoles of parenchyma cells due to breaking of the tonoplast where they presented higher stability. In both tissues, phenolic compounds were released from cell walls with increasing pressure and enhanced by cell wall ultrastructural modifications (100 MPa), rupture (350 MPa) and the rearrangement of microfibrillated cellulose (600 MPa). Prickly pears submitted to HHP presented advanced senescence marked by considerable ethylene increase and the gradual loss of CO2 production after 6 h. Cells were viable at 100 MPa by conserving intact cell membranes and after 24 h their respiration rates presented no significant differences compared to controls therefore indicating the possibility of synthesis of bioactive compound as a response to abiotic stress. We have proposed a new approach for analyzing the effects of HHP and have identified the storing of betalains in vesicles located in the cytoplasm of chlorenchyma cells for the first time. This study is the first to fathom the dynamic morphological changes and release mechanisms of bioactive compounds in vegetable cells subjected to HHP.

First description of extrafloral nectaries in Opuntia robusta (Cactaceae): Anatomy and ultrastructure.

To our knowledge, there are no studies about the structure and ecological function of extrafloral nectaries (EFNs) in Opuntia robusta. This is the first description of EFNs in O. robusta, where young spines have an interesting structure and a secreting function, which are different from EFNs described in other Cactaceae species. We used light, scanning-electron, and transmission-electron microscopy to examine morphology, anatomy, and ultrastructure of the secretory spines in areoles in female and hermaphrodite individuals of O. robusta. Young cladodes develop areoles with modified and secretory spines as EFNs only active during the early growth phase. EFNs are non-vascularized structures, with no stomata, that consist of a basal meristematic tissue, a middle elongation region, and an apical secretory cone formed by large globular epidermal cells, containing nectar and medullar elongated cells. We observed the presence of Golgi apparatus, vesicles and plastids in the medullar and sup-epidermal cells of the spine. We propose that the nectar is stored in the globular cells at the apex of the spine and secreted by breaking through the globular cells or by pores. We recorded a more frequent presence of ants on younger cladode sprouts producing young secreting spines: this result is parallel with the predictions of Optimal Defense Hypothesis, which states that younger plant organs should be better defended than older ones because their loss produces a higher fitness impairment. Although Diaz-Castelazo's hypothesis states that a more complex structure of EFNs correlates with their lower among-organs dispersion, comparing to less complex EFNs, non-vascularized structure of EFNs in O. robusta is not associated with their higher among-organs dispersion likened to O. stricta, which produces vascularized EFNs. We provide evidence that this characteristic is not a good taxonomic feature of Opuntia genus. Moreover, the comparison of EFNs of O. robusta and O. stricta suggests that the hypothesis of Diaz-Castelazo should be revised: it is rather a rule but not a law.

Effects of catalase on chloroplast arrangement in Opuntia streptacantha chlorenchyma cells under salt stress.

In arid and semiarid regions, low precipitation rates lead to soil salinity problems, which may limit plant establishment, growth, and survival. Herein, we investigated the NaCl stress effect on chlorophyll fluorescence, photosynthetic-pigments, movement and chloroplasts ultrastructure in chlorenchyma cells of Opuntia streptacantha cladodes. Cladodes segments were exposed to salt stress at 0, 100, 200, and 300 mM NaCl for 8, 16, and 24 h. The results showed that salt stress reduced chlorophyll content, F v /F m , ΦPSII, and qP values. Under the highest salt stress treatments, the chloroplasts were densely clumped toward the cell center and thylakoid membranes were notably affected. We analyzed the effect of exogenous catalase in salt-stressed cladode segments during 8, 16, and 24 h. The catalase application to salt-stressed cladodes counteracted the NaCl adverse effects, increasing the chlorophyll fluorescence parameters, photosynthetic-pigments, and avoided chloroplast clustering. Our results indicate that salt stress triggered the chloroplast clumping and affected the photosynthesis in O. streptacantha chlorenchyma cells. The exogenous catalase reverted the H2O2 accumulation and clustering of chloroplast, which led to an improvement of the photosynthetic efficiency. These data suggest that H2O2 detoxification by catalase is important to protect the chloroplast, thus conserving the photosynthetic activity in O. streptacantha under stress.

Micromorphology of cactus-pear (Opuntia ficus-indica (L.) Mill) cladodes based on scanning microscopies.

Cladode ultrastructural features of two prickly and two spineless Opuntia ficus-indica cultivars were examined using environmental scanning electron and atomic force microscopies. Observations focused on cladode as well as spine and glochid surface micromorphologies. Prickly cultivars were characterized by abundant cracked epicuticular wax deposits covering the cladode surface, with an amorphous structure as observed by AFM, while less abundant waxy plates were observed by ESEM on spineless cultivar cladodes. Further AFM observations allowed a rough granular and crystalloid epicuticular wax structure to be distinguished in spineless cultivars. Regarding spine micromorphology, prickly cultivars had strong persistent spines, observed by ESEM as a compact arrangement of oblong epidermal cells with a rough granular structure. However, deciduous spines in spineless cultivars had a broken transversely fissured epidermis covering a parallel arrangement of fibres. Through AFM, the deciduous spine surface presented an irregular hilly and smooth microrelief while persistent spines exhibited rough helical filamentous prints. ESEM and AFM studies of cladode surfaces from prickly and spineless cactus pear cultivars revealed valuable micro-morphological details that ought to be extended to a large number of O. ficus-indica cultivars.

Programmed cell death promotes male sterility in the functional dioecious Opuntia stenopetala (Cactaceae).

BACKGROUND AND AIMS: The sexual separation in dioecious species has interested biologists for decades; however, the cellular mechanism leading to unisexuality has been poorly understood. In this study, the cellular changes that lead to male sterility in the functionally dioecious cactus, Opuntia stenopetala, are described. METHODS: The spatial and temporal patterns of programmed cell death (PCD) were determined in the anthers of male and female flowers using scanning electron microscopy analysis and histological observations, focusing attention on the transition from bisexual to unisexual development. In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assays were used as an indicator of DNA fragmentation to corroborate PCD. KEY RESULTS: PCD was detected in anthers of both female and male flowers, but their patterns differed in time and space. Functionally male individuals developed viable pollen, and normal development involved PCD on each layer of the anther wall, which occurred progressively from the inner (tapetum) to the outer layer (epidermis). Conversely, functional female individuals aborted anthers by premature and displaced PCD. In anthers of female flowers, the first signs of PCD, such as a nucleus with irregular shape, fragmented and condensed chromatin, high vacuolization and condensed cytoplasm, occurred at the microspore mother cell stage. Later these features were observed simultaneously in all anther wall layers, connective tissue and filament. Neither pollen formation nor anther dehiscence was detected in female flowers of O. stenopetala due to total anther disruption. CONCLUSIONS: Temporal and spatial changes in the patterns of PCD are responsible for male sterility of female flowers in O. stenopetala. Male fertility requires the co-ordination of different events, which, when altered, can lead to male sterility and to functionally unisexual individuals. PCD could be a widespread mechanism in the determination of functionally dioecious species.

Effect of fungi and light on seed germination of three Opuntia species from semiarid lands of central Mexico.

Fungal attack under light reduces mechanical resistance of the testa of Opuntia seeds, making it easier for the embryo to emerge. However, the effect of fungi on Opuntia seed germination in darkness is unknown. We evaluated the combined effects of light and inoculation with Phoma medicaginis, Trichoderma harzianum, Trichoderma koningii, and Penicillium chrysogenum on germination of O. streptacantha, O. leucotricha, and O. robusta seeds, from central Mexico. We also evaluated the combined effects of seed age (2-, 3-, and 12-year-old seeds) and presence of fungi on the testa on O. streptacantha germination. All fungal species eroded the funicular envelope and promoted seed germination for O. leucotricha and O. streptacantha, but did more so in light than in darkness. For the latter species, younger seeds inoculated with fungi had lower germination than older ones. For O. robusta, we found that seeds inoculated with P. medicaginis and T. harzianum had similar germination in light and in darkness. Our results strongly indicate that deterioration of the testa by fungi is higher in light than in darkness.

Are fungi important for breaking seed dormancy in desert species? Experimental evidence in Opuntia streptacantha (Cactaceae).

Seeds of Opuntia spp. have physiological dormancy; they need a period of after-ripening to break dormancy, and the embryos have low growth potential. We evaluated the combined effects of seed age and presence of fungi on the testa on germination of Opuntia streptacantha, an abundant species in the Chihuahuan Desert (Mexico), assuming that older seeds have broken seed dormancy and fungi can reduce mechanical resistance to germination. In a preliminary experiment, we found no germination of 9-year-old (1998) and freshly collected (2007) seeds. However, we obtained 67% and 27% germination from 9-year-old and fresh non-sterilized seeds, respectively, and found fungi growing on the testa of all germinated seeds. Two fungal strains were isolated and identified using ribosomal internal transcribed spacer (ITS) sequence analysis: Penicillium chrysogenum and Phoma sp. In a second experiment, we inoculated seeds with strains of P. chrysogenum and Phoma sp., as well as Trichoderma koningii and binucleate Rhizoctonia (Gto17S2), to evaluate their ability to break seed dormancy. Seeds inoculated with P. chrysogenum, Phoma sp. and T. koningii had higher germination than controls for both seed ages, but germination was higher in older seeds. Scanning electron microscopy showed that these fungi eroded the funiculus, reducing its resistance. Binucleate Rhizoctonia did not lead to germination and controls had almost no germination. Our results strongly indicate that fungi are involved in breaking seed dormancy of O. streptacantha, and that the effect of fungi on seeds is species-specific.

Physicochemical characterization of nopal pads (Opuntia ficus indica) and dry vacuum nopal powders as a function of the maturation.

This paper presents the physicochemical and nutrimental characterization of fresh nopal (Opuntia ficus indica, Redonda variety) and nopal powder produced at different stages of development. Nopal powder was obtained by dry vacuum technique using 10(2) Torr and low temperature (40 degrees C). The results showed that the nutrimental and mineral composition of nopal changes as a function of the maturation as follow: The ash content increases from 18.41 for nopalitos (60 g of weight) to 23.24% (nopal pads 200 g); calcium content increases from 1.52 to 3.72%, while phosphorous exhibits an opposite trend: 0.43 to 0.27%, respectively. Calcium oxalate was determined by X-ray diffraction and SEM microscopy and quantified by using atomic absorption spectroscopy. Calcium oxalate decreases from 7.95 to 3.47 mg/g and the Ca/P ratio varies from 3.6 to 11. The soluble fibre decreases from 25.22 to 14.91%, while insoluble fibre increases from 29.87 to 41.65%. These results suggest that nopal could be an important source of minerals within the diets of people in Mexico and the rest of Latin America.

Morphological and secretory characterization of extrafloral nectaries in plants of coastal Veracruz, Mexico.

BACKGROUND AND AIMS: Morphological descriptions of the extrafloral nectaries (EFNs) of certain plant species are common in the literature, but they rarely relate morphology with histology, gland distribution and secretory attributes. In this study a morphological/secretory characterization of EFNs occurring on several plant species in a tropical coastal community is made and the implications of gland attributes discussed from a functional perspective. METHODS: The morphology and nectar secretion of the EFNs of 20 plant species are characterized through scanning electron microscopy, histochemical detection of reducing sugars (Fehling's reagent) and nectar volume/concentration estimates. KEY RESULTS: Sixty-five per cent of plant species in coastal communities had EFNs on vegetative structures and 35 % of species had glands on reproductive and vegetative organs. The Fabaceae is the plant family with the most species with EFNs and most diversity of gland morphologies. Four types of vascularized nectaries and four of glandular trichomes are described; sugar-secreting trichomes are characterized using Fehling's technique, and the first descriptions of unicellular and peltate trichomes functioning as EFNs are provided. Glands of ten plant species and six genera are described for the first time. Four plant species possess more than one morphological type of EFN. Eleven species have EFNs in more than one location or organ. More complex glands secrete more nectar, but are functionally homologous to the aggregations of numerous secretory trichomes on specific and valuable plant organs. CONCLUSION: Important diversity of EFN morphology was foundin the coastal plant community studied. Both vascularized and non-vascularized EFNs are observed in plants and, for the latter, previously non-existent morpho-secretory characterizations are provided with a methodological approach to study them. It is recommended that studies relating EFN attributes (i.e. morphology, distribution) with their differential visitation by insects (i.e. ants) and the cost of maintenance to the plants are carried out to understand the evolution of these glands.

Isolation and structure of D-xylans from pericarp seeds of Opuntia ficus-indica prickly pear fruits.

Xylans were isolated from the pericarp of prickly pear seeds of Opuntia ficus-indica (OFI) by alkaline extraction, fractionated by precipitation and purified. Six fractions were obtained and characterized by sugar analysis and NMR spectroscopy. They were assumed to be (4-O-methyl-D-glucurono)-D-xylans, with 4-O-alpha-D-glucopyranosyluronic acid groups linked at C-2 of a (1-->4)-beta-D-xylan. The sugar composition and the 1H and 13C NMR spectra showed that their chemical structures were very similar, but with different proportions of D-Xyl and 4-O-Me-D-GlcA. Our results showed that, on average, the water soluble xylans have one nonreducing terminal residue of 4-O-methyl-D-glucuronic acid for every 11 to 14 xylose units, whereas in the water non-soluble xylans, xylose units can varied from 18 to 65 residues for one nonreducing terminal residue of 4-O-methyl-D-glucuronic acid.