Histological assessment, anti-quorum sensing, and anti-biofilm activities of Dioon spinulosum extract: in vitro and in vivo approach.

Pseudomonas aeruginosa is an opportunistic bacterium causing several health problems and having many virulence factors like biofilm formation on different surfaces. There is a significant need to develop new antimicrobials due to the spreading resistance to the commonly used antibiotics, partly attributed to biofilm formation. Consequently, this study aimed to investigate the anti-biofilm and anti-quorum sensing activities of Dioon spinulosum, Dyer Ex Eichler extract (DSE), against Pseudomonas aeruginosa clinical isolates. DSE exhibited a reduction in the biofilm formation by P. aeruginosa isolates both in vitro and in vivo rat models. It also resulted in a decrease in cell surface hydrophobicity and exopolysaccharide quantity of P. aeruginosa isolates. Both bright field and scanning electron microscopes provided evidence for the inhibiting ability of DSE on biofilm formation. Moreover, it reduced violacein production by Chromobacterium violaceum (ATCC 12,472). It decreased the relative expression of 4 quorum sensing genes (lasI, lasR, rhlI, rhlR) and the biofilm gene (ndvB) using qRT-PCR. Furthermore, DSE presented a cytotoxic activity with IC50 of 4.36 ± 0.52 µg/ml against human skin fibroblast cell lines. For the first time, this study reports that DSE is a promising resource of anti-biofilm and anti-quorum sensing agents.

Arabinogalactan-proteins from non-coniferous gymnosperms have unusual structural features.

Arabinogalactan-proteins (AGPs), important signalling molecules of the plant cell wall, are structurally extensively investigated in angiosperms, but information on AGPs in gymnosperms is still limited. We characterized AGPs from the gymnosperms Ginkgo biloba, Ephedra distachya, Encephalartos longifolius and Cycas revoluta. The protein contents are comparable to that of angiosperm AGPs. Hydroxyproline is the site of linking the carbohydrate part and was detected in all AGPs with highest concentration in Cycas AGP (1.1 % of the AGP). Interestingly, with the exception of Cycas, all AGPs contained the monosaccharide 3-O-methylrhamnose not present in angiosperm polysaccharides. The carbohydrate moieties of Cycas and Ephredra showed the main components 1,3,6-linked galactose and terminal arabinose typical of angiosperm AGPs, whereas that of Ginkgo AGP was unique with 1,4-linked galactose as dominant structural element. Bioinformatic search for glycosyltransferases in Ginkgo genome also revealed a lower number of galactosyltransferases responsible for biosynthesis of the 1,3-Gal/1,6-Gal AGP backbone.

Embryo cell wall properties in relation to development and desiccation in the recalcitrant-seeded Encephalartos natalensis (Zamiaceae) Dyer and Verdoorn.

Plant cell walls are dynamic entities that may change with development, differ between plant species and tissue type and play an important role in responses to various stresses. In this regard, the present investigation employed immunocytochemistry to determine wall composition and possible changes during development of immature and mature embryos of the recalcitrant-seeded cycad Encephalartos natalensis. Fluorescent and gold markers, together with cryo-scanning and transmission electron microscopy (TEM) were also used to analyse potential changes in the cell walls of mature embryos upon desiccation. Immature cell walls were characterised by low- and high methyl-esterified epitopes of pectin, rhamnogalacturonan-associated arabinan, and the hemicellulose xyloglucan. Arabinogalactan protein recognised by the LM2 antibody, along with rhamnogalacturonan-associated galactan and the hemicellulose xylan, were not positively localised using immunological probes, suggesting that the cell walls of the embryo of E. natalensis do not possess these epitopes. Interestingly, mature embryos appeared to be identical to immature ones with respect to the cell wall components investigated, implying that these may not change during the protracted post-shedding embryogenesis of this species. Drying appeared to induce some degree of cell wall folding in mature embryos, although this was limited by the abundant amyloplasts, which filled the cytomatrical space. Folding, however, was correlated with relatively high levels of wall plasticisers typified by arabinose polymers. From the results of this study, it is proposed that the embryo cell walls of E. natalensis are constitutively prepared for the flexibility required during cell growth and expansion, which may also facilitate the moderate cell wall folding observed in mature embryos upon drying. This, together with the abundant occurrence of amyloplasts in the cytomatrix, may provide sufficient mechanical stabilisation if water is lost, even though the seeds of this species are highly desiccation-sensitive.

Can stable isotopes and radiocarbon dating provide a forensic solution for curbing illegal harvesting of threatened cycads?

Cycads in South Africa are facing an extinction crisis due to the illegal extraction of plants from the wild. Proving wild origin of suspect ex situ cycads to the satisfaction of a court of law is difficult, limiting law enforcement efforts. We investigated the feasibility of using multiple stable isotopes to identify specimens removed from the wild. Relocated and wild specimens from two species in the African genus Encephalartos (E. lebomboensis and E. arenarius) were sampled. (14) C analysis indicated that a ± 30-year chronology could be reliably obtained from the cycads. For E. arenarius, pre-relocation tissue was consistent with a wild origin, whereas tissue grown post-relocation was isotopically distinct from the wild for (87) Sr/(86) Sr and δ(15) N. For E. lebomboensis, δ(34) S, δ(18) O, and (87) Sr/(86) Sr were different between relocated and control plants, consistent with the >30 years since relocation. Our findings demonstrate the potential for a forensic isotope approach to identify illegal ex situ cycads.

Relative seed and fruit toxicity of the Australian cycads Macrozamia miquelii and Cycas ophiolitica: further evidence for a megafaunal seed dispersal syndrome in cycads, and its possible antiquity.

An apparent contradiction in the ecology of cycad plants is that their seeds are known to be highly poisonous, and yet they seem well adapted for seed dispersal by animals, as shown by their visually conspicuous seed cones and large seeds presented within a brightly colored fleshy "fruit" of sarcotesta. We tested if this sarcotesta could function as a reward for cycad seed dispersal fauna, by establishing if the toxic compound cycasin, known from the seeds, is absent from the sarcotesta. The Australian cycads Macrozamia miquelii and Cycas ophiolitica were tested (N = 10 individuals per species) using gas chromatography / mass spectrometry. Cycasin was detected at 0.34 % (fresh weight) in seed endosperm of M. miquelii and 0.28 % (fresh weight) in seed endosperm of C. ophiolitica. Cycasin was absent from the sarcotesta of the same propagules (none detected in the case of M. miquelii, and trace quantities detected in sarcotesta of only four of the ten C. ophiolitica propagules). This laboratory finding was supported by field observations of native animals eating the sarcotesta of these cycads but discarding the toxic seed intact. These results suggest cycads are adapted for dispersal fauna capable of swallowing the large, heavy propagules whole, digesting the non-toxic sarcotesta flesh internally, and then voiding the toxic seed intact. Megafauna species such as extant emus or cassowaries, or extinct Pleistocene megafauna such as Genyornis, are plausible candidates for such dispersal. Cycads are an ancient lineage, and the possible antiquity of their megafaunal seed dispersal adaptations are discussed.

Leaf traits and herbivory levels in a tropical gymnosperm, Zamia stevensonii (Zamiaceae).

PREMISE OF THE STUDY: Slow-growing understory cycads invest heavily in defenses to protect the few leaves they produce annually. The Neotropical cycad Zamia stevensonii has chemical and mechanical barriers against insect herbivores. Mechanical barriers, such as leaf toughness, can be established only after the leaf has expanded. Therefore, chemical defenses may be important during leaf expansion. How changes in leaf traits affect the feeding activity of cycad specialist insects is unknown. We investigated leaf defenses and incidence of specialist herbivores on Z. stevensonii during the first year after leaf flush. METHODS: Herbivore incidence, leaf production, and leaf traits that might affect herbivory-including leaf age, lamina thickness, resistance-to-fracture, work-to-fracture, trichome density, and chlorophyll, water, and toxic azoxyglycoside (AZG) content-were measured throughout leaf development. Principal component analysis and generalized linear models identified characteristics that may explain herbivore incidence. KEY RESULTS: Synchronized leaf development in Z. stevensonii is characterized by quick leaf expansion and delayed greening. Specialist herbivores feed on leaves between 10 and 100 d after flush and damage ∼37% of all leaflets produced. Young leaves are protected by AZGs, but these defenses rapidly decrease as leaves expand. Leaves older than 100 d are protected by toughness. CONCLUSIONS: Because AZG concentrations drop before leaves become sufficiently tough, there is a vulnerable period during which leaves are susceptible to herbivory by specialist insects. This slow-growing gymnosperm invests heavily in constitutive defenses against highly specialized herbivores, underlining the convergence in defensive syndromes by major plant lineages.

Patterns of odour emission, thermogenesis and pollinator activity in cones of an African cycad: what mechanisms apply?

BACKGROUND AND AIMS: Ontogenetic patterns of odour emissions and heating associated with plant reproductive structures may have profound effects on insect behaviour, and consequently on pollination. In some cycads, notably Macrozamia, temporal changes in emission of specific odour compounds and temperature have been interpreted as a 'push-pull' interaction in which pollinators are either attracted or repelled according to the concentration of the emitted volatiles. To establish which mechanisms occur in the large Encephalartos cycad clade, the temporal patterns of volatile emissions, heating and pollinator activity of cones of Encephalartos villosus in the Eastern Cape (EC) and KwaZulu Natal (KZN) of South Africa were investigated. METHODS AND KEY RESULTS: Gas chromatography-mass spectrometry (GC-MS) analyses of Encephalartos villosus cone volatiles showed that emissions, dominated by eucalyptol and 2-isopropyl-3-methoxypyrazine in EC populations and (3E)-1,3-octadiene and (3E,5Z)-1,3,5-octatriene in the KZN populations, varied across developmental stages but did not vary significantly on a daily cycle. Heating in male cones was higher at dehiscence than during pre- and post-dehiscence, and reached a maximum at about 1830 h when temperatures were between 7·0 and 12·0 °C above ambient. Daily heating of female cones was less pronounced and reached a maximum at about 1345 h when it was on average between 0·9 and 3·0 °C above ambient. Insect abundance on male cones was higher at dehiscence than at the other stages and significantly higher in the afternoon than in the morning and evening. CONCLUSIONS: There are pronounced developmental changes in volatile emissions and heating in E. villosus cones, as well as strong daily changes in thermogenesis. Daily patterns of volatile emissions and pollinator abundance in E. villosus are different from those observed in some Macrozamia cycads and not consistent with the push-pull pattern as periods of peak odour emission do not coincide with mass exodus of insects from male cones.

Variation in the chemical composition of cone volatiles within the African cycad genus Encephalartos.

Volatiles play a key role in attraction of pollinators to cycad cones, but the extent to which volatile chemistry varies among cycad species is still poorly documented. Volatile composition of male and female cones of nineteen African cycad species (Encephalartos; Zamiaceae) was analysed using headspace technique and gas chromatography-mass spectrometry (GC-MS). A total of 152 compounds were identified among the species included in this study, the most common of which were monoterpenes, nitrogen-containing compounds and unsaturated hydrocarbons. Male and female cones emitted similar volatile compounds which varied in relative amounts with two unsaturated hydrocarbons (3E)-1,3-octadiene and (3E,5Z)-1,3,5-octatriene present in the volatile profile of most species. In a multivariate analysis of volatile profiles using non-metric multidimensional scaling (NMDS), a number of species clusters were identified according to shared emission of unsaturated hydrocarbons, pyrazines, benzenoids, aldehydes, alkanes and terpenoids. In comparison, terpenoids are common in Zamia and dominant in Macrozamia species (both in the family Zamiaceae) while benzenoids, esters, and alcohols are dominant in Cycas (Cycadaceae) and in Stangeria (Stangeriaceae). It is likely that volatile variation among Encephalartos species reflects both phylogeny and adaptations to specific beetle pollinators.

Two genera of Aulacoscelinae beetles reflexively bleed azoxyglycosides found in their host cycads.

Aulacoscelinae beetles have an ancient relationship with cycads (Cycadophyta: Zamiaceae), which contain highly toxic azoxyglycoside (AZG) compounds. How these "primitive" leaf beetles deal with such host-derived compounds remains largely unknown. Collections were made of adult Aulacoscelis appendiculata from Zamia cf. elegantissima in Panama, A. vogti from Dioon edule in Mexico, and Janbechynea paradoxa from Zamia boliviana in Bolivia. Total AZG levels were quantified in both cycad leaves and adult beetles by high performance liquid chromatography (HPLC). On average, cycad leaves contained between 0.5-0.8% AZG (frozen weight, FW), while adult beetles feeding on the same leaves contained even higher levels of the compounds (average 0.9-1.5% FW). High AZG levels were isolated from reflex bleeding secreted at the leg joints when beetles were disturbed. Nuclear magnetic resonance and mass spectroscopy identified two AZGs, cycasin and macrozamin, in the reflex bleeding; this is the first account of potentially plant-derived compounds in secretions of the Aulacoscelinae. These data as well as the basal phylogenetic position of the Aulacoscelinae suggest that sequestration of plant secondary metabolites appeared early in leaf beetle evolution.