Neusa Martins

Micropropagation and conservation of endangered species Plantago algarbiensis and P. almogravensis

Plantago algarbiensis and P. almogravensis are endemic Al tolerant species from the Western-centre of the Algarve region (South of Portugal) and Portuguese Southwest coast, respectively, which are in risk of global extinction. The aim of this work was to establish an efficient protocol to in vitro propagate these species using shoots obtained from in vitro germinated seeds. The best results in terms of multiplication response were afforded in Murashige and Skoog’s (MS) medium supplemented with 6-benzyladenine (8.5 and 9.2 shoots per explant in P. algarbiensis and P. almogravensis, respectively). Shoots of both species showed a great rooting capacity (100 and 80 % for P. algarbiensis and P. almogravensis, respectively) that was not significantly influenced by the concentration of MS macronutrients or auxins. Plants were acclimatized to ex vitro conditions, exhibited normal development (survival rate of 95 and 80 % in P. algarbiensis and P. almogravensis, respectively), and were successfully reintroduced in their natural habitat.

The influence of low pH on in vitro growth and biochemical parameters of Plantago almogravensis and P. algarbiensis

The effects of low medium pH (4.50, 5.00 and 5.75) on in vitro growth and on several biochemical parameters (lipid peroxidation, proline and carbohydrate content, antioxidant enzymes activities and total soluble protein) of Plantago almogravensis and P. algarbiensis micropropagated shoots were investigated. Overall, it was observed that medium pH did not affect in vitro proliferation and rooting. Interestingly, cultures of both species modify the initial pH value to the same final value. Results have shown that the lowest pH tested induced an increase in the level of lipid peroxidation in roots of both species and in shoots of P. algarbiensis, indicating plasma membrane damage. An accumulation of carbohydrates was observed in roots of P. almogravensis cultured in pH 4.50 and 5.00. It was observed a slight response of the enzymatic system to medium pH, particularly in P. almogravensis. Based on the results obtained we can conclude that Plantago species are apt to grow in vitro in medium with pH values much lower than the usually used in tissue culture, which is in agreement with the fact that both species colonize acid soils.

Changes on organic acid secretion and accumulation in Plantago almogravensis Franco and Plantago algarbiensis Samp. under aluminum stress

We investigated the effect of Al (400μM) on organic acids secretion, accumulation and metabolism in Plantago almogravensis Franco and Plantago algarbiensis Samp. Al induced a significant reduction on root elongation only in P. algarbiensis. Both species accumulated considerable amounts of Al (>120μgg(-1)) in their tissues, roots exhibiting the highest contents (>900μgg(-1)). Al stimulated malonic acid secretion in P. algarbiensis, while citric, succinic and malic acids were secreted by P. almogravensis. Moreover, Al uptake was accompanied by substantial increases of citric, oxalic, malonic and fumaric acids contents in the plantlets of either species. Overall, the acid metabolizing enzymes were not directly involved in the Al induced organic acid secretion and accumulation. Our data suggest that Al detoxification in P. almogravensis implies both secretion of organic acids from roots and tolerance to high Al tissue concentrations, while in P. algarbiensis only the tolerance mechanism seems to be involved.

Metabolism and aluminum accumulation in Plantago almogravensis and P. algarbiensis in response to low pH and aluminum stress

We investigated the impact of low pH and aluminum on the metabolism and capacity for Al accumulation in shoots of the plantain species Plantago algarbiensis and P. almogravensis. We found that increasing the concentration of Al in the medium increased accumulation of it in the shoots of both plants (although more in P. almogravensis than in P. algarbiensis). The presence of Al in the medium induced proline and saccharide synthesis in P. almogravensis without affecting lipid peroxidation, but increased proline synthesis and lipid peroxidation in P. algarbiensis without affecting the saccharide content. Lipid peroxidation in P. algarbiensis was also enhanced at pH 4.0. The activity of antioxidant enzymes was increased as a response to low pH and Al in both species. Our data indicate that both species can accumulate high levels of Al but they have different sensitivities to low pH and/or the presence of Al in the growth medium.

Differences in Al tolerance between Plantago algarbiensis and P. almogravensis reflect their ability to respond to oxidative stress

We evaluated the impact of low pH and aluminum (Al) on the leaves and roots of Plantago almogravensis Franco and Plantago algarbiensis Samp., focusing on energy partitioning in photosystem II, H2O2 levels, lipid peroxidation, electrolyte leakage (EL), protein oxidation, total soluble protein content and antioxidant enzyme activities. In both species, Al triggered more changes in oxidative metabolism than low pH alone, particularly in the roots. We found that Al increased the levels of H2O2 in P.algarbiensis roots, but reduced the levels of H2O2 in P. almogravensis leaves and roots. Neither low pH nor Al affected the spatial heterogeneity of chlorophyll fluorescence, the maximum photochemical efficiency of PSII (Fv/Fm), the actual quantum efficiency of PSII (ϕPSII) or the quantum yields of regulated (ϕNPQ) and nonregulated (ϕNO) energy dissipation, and there was no significant change in total soluble protein content and EL. In P. algarbiensis, Al increased the carbonyl content and the activities of superoxide dismutase (SOD) and catalase (CAT) in the roots, and also CAT, ascorbate peroxidase and guaiacol peroxidase activities in the leaves. In P. almogravensis, Al reduced the level of malondialdehyde in the roots as well as SOD activity in the leaves and roots. We found that P. almogravensis plantlets could manage the oxidative stress caused by low pH and Al, whereas the P.algarbiensis antioxidant system was unable to suppress Al toxicity completely, leading to the accumulation of H2O2 and consequential protein oxidation in the roots.

Physiological traits and oxidative stress markers during acclimatization of micropropagated plants from two endangered Plantago species : P. algarbiensis Samp. and P. almogravensis Franco

Plantago algarbiensis Samp. and Plantago almogravensis Franco are species endemic to Portugal at risk of global extinction. The aim of this study was to investigate the ex vitro performance of micropropagated P. algarbiensis and P. almogravensis plants in terms of survival, relative water content (RWC), photosynthetic pigment contents, H2O2 accumulation, activities of superoxide dismutase (SOD) and catalase (CAT), lipid peroxidation, and soluble protein content, in comparison with wild-grown plants. Relatively high survival rates and RWC values during the acclimatization process were observed for both species. In P. algarbiensis, the pigment content increased when plantlets were transferred to ex vitro conditions, indicating enhanced light absorption capacity. No significant alterations in H2O2 content, CAT activity, or lipid peroxidation level were observed during acclimatization, but the protein content decreased in plants at the end of the growth chamber and greenhouse stages. When P. almogravensis plantlets were transferred to the ex vitro environment, decreases in the H2O2 content were observed that correlated to increased CAT activity and SOD maintenance, which lead to decreased lipid peroxidation and protein content. It was concluded that micropropagated P. algarbiensis and P. almogravensis plants were able to manage the oxidative stress induced by the in vitro environment and to perform well under ex vitro conditions.

Molecular instability induced by aluminum stress in Plantago species.

Aluminum (Al) is one of the most abundant metals on earths crust and Al toxicity represents one of the major factors that limit plant growth and productivity in acid soils (with a pH≤5.0). In this study the mutagenic/genotoxic effects of Al were evaluated in roots and leaves of two Plantago, species, Plantago almogravensis and Plantago lagopus, using ISSRs markers. Both species were exposed to 400 μM Al during 7 and 21 days. Ten ISSR primers produced polymorphic bands. In P. almogravensis, a total of 257 and 258 bands in roots and 255 and 265 bands in leaves were produced in the presence and absence of Al, respectively. In P. lagopus were produced 279 and 278 a total bands in roots and 275 and 274 bands in leaves, under the same conditions. The changes in ISSR profiles after Al treatment were considered as gain and/or loss of bands compared with the controls. The results suggest that changes in genomic template stability (GTS) could be detected with ISSR profiles. This molecular marker proved to be a good tool to detect the effects of Al on DNA profiles. It seems that Al did not interfere significantly with DNA integrity in both species but generated less ISSR stability in P. almogravensis than in P. lagopus. The results confirm the tolerance of P. almogravensis and suggest the same behavior of P. lagopus. Although further studies are required for confirmation the Al tolerance behavior of P. lagopus, a potential application for phytoremediation can be also considered due its wide distribution.

Interactions of daylength, temperature and nutrients affect thresholds for life stage transitions in the kelp Laminaria digitata (Phaeophyceae)

Abstract Kelp beds worldwide are under pressure from ongoing climate and environmental change. Along European coastlines increases in seawater temperature and changes in nutrient conditions occur where upwelling events are disrupted and also along eutrophicated coasts. In addition, seaweed responses to change may interact with seasonal daylength cycles. We performed a factorial experiment to examine the combined effects of seawater temperatures, nutrient regimes and photoperiod (long and short days) in order to better understand how latitudinal or seasonal differences in daylengths affect the sensitivity of transient microscopic kelp stages of Laminaria digitata from the North Sea to warming and eutrophication. While the optimal temperature range for vegetative gametophyte growth was 10°C–18°C under long summer photoperiod conditions, gametogenesis was induced at lower temperatures between 5°C and 15°C, with maximum sporophyte development under long photoperiods and enriched nutrient regimes, which represent local late spring conditions. Although gametogenesis was fastest at 10°C–15°C, sporophyte recruitment was highest at 5°C. As these particular early life cycle processes in L. digitata have different temperature optima, this may drive the seasonal cycle of recruitment in the field. Increasing summer temperatures due to global warming will increase gametophyte size due to enhanced vegetative growth and inhibition of gametogenesis. This will probably lead to delayed but enhanced recruitment of new sporophytes under cooler autumn to spring conditions over a wide geographical scale, preventing the formation of juvenile sporophytes under stressful summer conditions and possibly changing annual recruitment patterns.

Cryptic diversity, geographical endemism and allopolyploidy in NE Pacific seaweeds

BackgroundMolecular markers are revealing a much more diverse and evolutionarily complex picture of marine biodiversity than previously anticipated. Cryptic and/or endemic marine species are continually being found throughout the world oceans, predominantly in inconspicuous tropical groups but also in larger, canopy-forming taxa from well studied temperate regions. Interspecific hybridization has also been found to be prevalent in many marine groups, for instance within dense congeneric assemblages, with introgressive gene-flow being the most common outcome. Here, using a congeneric phylogeographic approach, we investigated two monotypic and geographically complementary sister genera of north-east Pacific intertidal seaweeds (Hesperophycus and Pelvetiopsis), for which preliminary molecular tests revealed unexpected conflicts consistent with unrecognized cryptic diversity and hybridization.ResultsThe three recovered mtDNA clades did not match a priori species delimitations. H. californicus was congruent, whereas widespread P. limitata encompassed two additional narrow-endemic species from California - P. arborescens (here genetically confirmed) and P. hybrida sp. nov. The congruence between the genotypic clusters and the mtDNA clades was absolute. Fixed heterozygosity was apparent in a high proportion of loci in P. limitata and P. hybrida, with genetic analyses showing that the latter was composed of both H. californicus and P. arborescens genomes. All four inferred species could be distinguished based on their general morphology.ConclusionsThis study confirmed additional diversity and reticulation within NE Pacific Hesperophycus/Pelvetiopsis, including the validity of the much endangered, modern climatic relict P. arborescens, and the identification of a new, stable allopolyploid species (P. hybrida) with clearly discernable ancestry (♀ H. californicus x ♂ P. arborescens), morphology, and geographical distribution. Allopolyploid speciation is otherwise completely unknown in brown seaweeds, and its unique occurrence within this genus (P. limitata possibly representing a second example) remains enigmatic. The taxonomic separation of Hesperophycus and Pelvetiopsis is not supported and the genera should be synonymized; we retain only the latter. The transitional coastline between Point Conception and Monterey Bay represented a diversity hotspot for the genus and the likely sites of extraordinary evolutionary events of allopolyploid speciation at sympatric range contact zones. This study pinpoints how much diversity (and evolutionary processes) potentially remains undiscovered even on a conspicuous seaweed genus from the well-studied Californian intertidal shores let alone in other, less studied marine groups and regions/depths.