Secretory structures in plants: Lessons from the Plumbaginaceae on their origin, evolution and roles in stress tolerance.

The Plumbaginaceae (non-core Caryophyllales) is a family well known for species adapted to a wide range of arid and saline habitats. Of its salt-tolerant species, at least 45 are in the genus Limonium; two in each of Aegialitis, Limoniastrum and Myriolimon, and one each in Psylliostachys, Armeria, Ceratostigma, Goniolimon and Plumbago. All the halophytic members of the family have salt glands and salt glands are also common in the closely related Tamaricaceae and Frankeniaceae. The halophytic species of the three families can secrete a range of ions (Na+ , K+ , Ca2+ , Mg2+ , Cl- , HCO3- , SO42- ) and other elements (As, Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn). Salt glands are, however, absent in salt-tolerant members of the sister family Polygonaceae. We describe the structure of the salt glands in the three families and consider whether glands might have arisen as a means to avoid the toxicity of Na+ and/or Cl- or to regulate Ca2+ concentrations with the leaves. We conclude that the establishment of lineages with salt glands took place after the split between the Polygonaceae and its sister group the Plumbaginaceae.

Harnessing sediments of coastal aquaculture ponds through technosols construction for halophyte cultivation using saline water irrigation.

The Mediterranean aquaculture has been developed mostly in brackish environment in inactive coastal salt production areas. This study aims to utilise Technosols made with aquaculture sediments for Limonium algarvense Erben cultivation. This species that has nutraceutical potential thrives in halophilic environments in the southwest of the Iberian Peninsula and in Morocco. A microcosm assay was set up with plants grown in bottom sediments (C+), commercial substrate (C-), and Technosols with amendments mixture application at 180 g/kg (Tec180) or at 360 g/kg (Tec360). These plants were irrigated with saline (assay 1) and/or with deionised water (assay 2). The bottom pond sediments, coffee wastes and the estuarine water were evaluated for diverse physicochemical parameters. Plant growth was characterised through a combined methodology using morphometric, SEM and physiological analysis. The Technosols were constructed with bottom sediments and a mixture of organic wastes used as amendments. Results revealed that the bottom sediments had low pH 3.2, Corg and extractable P and K contents, and high electroconductivity (EC) and N-NH4 concentration. The estuarine water had a neutral pH, high EC and high Cl-, HCO3-, Na+, Mg2+ and Ca2+ but low N-NO3- content. The Technosols showed a significant increase of pH, Corg, K and P and a decrease in N-NH4 and EC in comparison with sediments. Principal component analysis separated the different experiments in three groups: C-, A1 and A2 assays. The C- was highly correlated with Corg, P, K, N-NO3 parameters and total ascorbate. The A1 assay showed a strong association with Na, Ca and EC parameters, whereas the A2 assay presented a strongly correlation with plant growth. Plants from Technosols had greater development when irrigated with deionised water than under salty irrigation as opposed to plants cultivated in unamend sediments. In conclusion, these results support that highly saline sediments could be valorised through Technosols construction to cultivate plants with saline water, with potential application in the agro-food and pharmaceutical industry.

Phylogeography and modes of reproduction in diploid and tetraploid halophytes of Limonium species (Plumbaginaceae): evidence for a pattern of geographical parthenogenesis.

BACKGROUND AND AIMS: The genus Limonium (Plumbaginaceae) has long been recognized to have sexual and apomictic (asexual seed formation) modes of reproduction. This study aimed to elucidate phylogeographical patterns and modes of reproduction in diploid and tetraploid Limonium species, namely three putative sexual diploid species with morphological affinities (L. nydeggeri, L. ovalifolium, L. lanceolatum) and three related, probably apomict tetraploid species (L. binervosum, L. dodartii, L. multiflorum). METHODS: cpDNA diversity and differentiation between natural populations of the species were investigated using two chloroplast sequence regions (trnL intron and trnL-trnF intergenic spacer). Floral heteromorphies, ovule cytoembryological analyses and pollination and crossing tests were performed in representative species of each ploidy group, namely diploid L. ovalifolium and tetraploid L. multiflorum, using plants from greenhouse collections. KEY RESULTS AND CONCLUSIONS: Genetic analyses showed that diploid species have a higher haplotype diversity and a higher number of unique (endemic) haplotypes than tetraploid species. Network analysis revealed correlations between cpDNA haplotype distribution and ploidy groups, species groups and geographical origin, and haplotype sharing within and among species with distinct ploidy levels. Reproductive biology analyses showed that diploid L. ovalifolium mainly forms meiotically reduced tetrasporic embryo sacs of Gagea ova, Adoxa and Drusa types. Limonium multiflorum, however, has only unreduced, diplosporic (apomictic) embryo sacs of Rudbeckia type, and autonomous apomictic development seems to occur. Taken together, the findings provide evidence of a pattern of 'geographical parthenogenesis' in which quaternary climatic oscillations appear to be involved in the geographical patterns of coastal diploid and tetraploid Limonium species.

Acorn selection by the wood mouse Apodemus sylvaticus: a semi-controlled experiment in a Mediterranean environment.

Fruits are highly important food resources for mammals in Mediterranean Europe, and due to the dominance of oaks (Quercus sp.), acorns are among those used by a vast array of species, including rodents. The metabolic yield of acorn intake may determine a selection pattern: preference for fat, carbohydrate, and consequently energy-rich fruits; or avoidance of fruits containing high concentrations of secondary chemical compounds (e.g., tannic acid). We studied the acorn feeding selection pattern of wood mice (Apodemus sylvaticus) inhabiting a mixed oak woodland, southwest Portugal, using an experiment conducted in an open-air enclosure. We tested which variables associated with the wood mouse (e.g., sex) and acorns (e.g., size and nutrient content) from three oak species (holm Q. rotundifolia, Portuguese Q. faginea and cork Q. suber oak) could be constraining acorn consumption. Our results indicate that wood mice are selecting acorns of the most common oak species (Q. suber), probably due to their previous familiarization with the fruit due to its dominance in the ecosystem but probably also because its chemical characteristics (sugar contents). Rodent gender and acorn morphology (width) are also influential, with females more prone to consume acorns with smaller width, probably due to handling limitation. This selective behaviour may have consequences for dispersion and natural regeneration of the different oak species.

Male fertility versus sterility, cytotype, and DNA quantitative variation in seed production in diploid and tetraploid sea lavenders (Limonium sp., Plumbaginaceae) reveal diversity in reproduction modes.

The genus Limonium Miller, a complex taxonomic group, comprises annuals and perennials that can produce sexual and/or asexual seeds (apomixis). In this study, we used diverse cytogenetic and cytometric approaches to analyze male sporogenesis and gametogenesis for characterizing male reproductive output on seed production in Limonium ovalifolium and Limonium multiflorum. We showed here that the first species is mostly composed of diploid cytotypes with 2n = 16 chromosomes and the latter species by tetraploid cytotypes with 2n = 32, 34, 35, 36 chromosomes and had a genome roughly twice as big as the former one. In both species, euploid and aneuploid cytotypes with large metacentric chromosomes having decondensed interstitial sites were found within and among populations, possibly involved in chromosomal reconstructions. L. ovalifolium diploids showed regular meiosis resulting in normal tetrads, while diverse chromosome pairing and segregation irregularities leading to the formation of abnormal meiotic products are found in balanced and non-balanced L. multiflorum tetraploids. Before anther dehiscence, the characteristic unicellular, bicellular, or tricellular pollen grains showing the typical Limonium micro- or macro-reticulate exine ornamentation patterns were observed in L. ovalifolium using scanning electron microscopy. Most of these grains were viable and able to produce pollen tubes in vitro. In both balanced and unbalanced L. multiflorum tetraploids, microspores only developed until the "ring-vacuolate stage" with a collapsed morphology without the typical exine patterns, pointing to a sporophytic defect. These microspores were unviable and therefore never germinated in vitro. L. ovalifolium individuals presented larger pollen grains than those of L. multiflorum, indicating that pollen size and ploidy levels are not correlated in the Limonium system. Cytohistological studies in mature seeds from both species revealed that an embryo and a residual endosperm were present in each seed. Flow cytometric seed screens using such mature seeds showed quantitative variations in seeds ploidy level. It is concluded that male function seems to play an important role in the reproduction modes of Limonium diploids and tetraploids.

Antioxidant and antimycotic activities of two native lavandula species from portugal.

The antioxidant and antimycotic activities of the essential oils and extracts of two native Portuguese Lavandula species, L. stoechas subsp. luisieri and L. pedunculata, were evaluated by in vitro assays. The total phenolics and flavonoids content were also determined. The antioxidant potential was assessed through DPPH radical scavenging, inhibition of lipid peroxidation (ILP), and DNA protection assays. All samples displayed a high DPPH scavenging activity, some of them showing concentration dependence. The majority of the samples were also able to inhibit lipid peroxidation. A strong correlation was observed between the results of DPPH and ILP assays and the flavonoids content of the samples. In the DNA protection assay, all the extracts were able to preserve DNA integrity. The antimycotic activity was performed against twelve fungi belonging to Basidiomycota and Ascomycota Divisions. L. stoechas subsp. luisieri exhibited the broadest activity spectra. L. pedunculata extracts were active against five fungi. Cryptococcus neoformans was the most sensitive, being inhibited by all the extracts. Our results led to the conclusion that L. stoechas subsp. luisieri and L. pedunculata can be useful as new sources of natural antioxidants and antimycotic agents, providing a possible valorization of the existing biodiversity and resources of Portuguese flora.

Flavonoids of an extract of Pterospartum tridentatum showing endothelial protection against oxidative injury.

Pterospartum tridentatum is a Leguminosae that grows spontaneously in Portugal. The flowers are used in popular medicine for the treatment of throat irritation conditions and in herbal mixtures for diabetes. Diabetic vascular complications are due, among other reasons, to increased oxidative stress and for that reason antioxidants are believed to be beneficial for the diabetic patient. The flower water extract of this herbal drug showed dose-dependent protective effect of cultured human endothelial cells against oxidative injury induced by H2O2, at concentrations > or =0.3 mg/ml. This water extract, after liquid-liquid and chromatographic fractionation afforded one new isoflavone (5,5'-dihydroxy-3'-metoxi-isoflavone-7-O-beta-glucoside) and three other known isoflavones (prunetin, genistin and sissotrin). The structural characterisation of isolated compounds was achieved by UV, NMR and MS analysis. The flavonol glycoside isoquercitrin was also identified in the extract by HPLC analysis. Isoquercitrin is one of the active antioxidant principles of the extract since it showed dose-dependent protective effect against oxidative injury at concentrations > or =0.3 mM. Isoflavones were inactive at the same concentrations. The results suggest that the water extract of this herbal drug may prevent or reduce the development of diabetic vascular complications.