Luis Balaguer

A reappraisal of the use of DMSO for the extraction and determination of chlorophylls a and b in lichens and higher plants

Abstract The use of DMSO for the extraction and determination of chlorophylls a and b in lichens and higher plants was reevaluated. Because of differences between the absorption spectra of pure chlorophylls a and b in DMSO and 80% acetone, formulae to calculate the individual concentrations of chlorophyll a, chlorophyll b and total (a + b) chlorophyll in pigment extracts were redetermined for specific use with DMSO. In lichens, the problem of chlorophyll degradation resulting from the presence of acidic lichen substances was specifically addressed. Repeated washing of thalli with carbonate-saturated 100% acetone followed by extraction in DMSO containing PVPi minimized the conversion of chlorophylls to phaeophytin during extraction of chlorophylls from lichens for which the content of lichen substances was characterized. In lichens containing significant quantities of acidic compounds, the modified DMSO assay proved superior to 80% acetone for the extraction and determination of chlorophyll a and b concentrations. In a range of higher plants, determinations of chlorophyll a and b concentrations were virtually identical when the modified DMSO assay was compared with the traditional method of chlorophyll extraction using 80% acetone. Moreover, DMSO extracts could be cold-stored for up to 7 days with no significant loss of chlorophylls a or b, or changes in the a/b ratio. Potential eco-physiological applications of the modified DMSO assay, which eliminates the necessity for grinding plant material and centrifuging plant extracts, are discussed.

Ecophysiological significance of chlorophyll loss and reduced photochemical efficiency under extreme aridity in Stipa tenacissima L.

Stipa tenacissima L., a perennial tussock grass widely found in semi-arid environments of the Iberian Peninsula and North Africa, is subjected to multiple stresses during the extreme summer conditions of south-east Spain. We characterised the photoprotective mechanisms of S. tenacissima during the transition from spring to summer and autumn. S. tenacissima experienced a marked water deficit (Ψ{ pd} < -8.4 MPa) and the complete suppression of CO2 assimilation in August, associated with a 72% reduction of maximal photochemical efficiency of PSII (F{ v}/F{ m}). These reduced F{ v}/F{ m} values were related to the pre-dawn maintenance of high levels of epoxidized forms of xanthophyll-cycle pigments (DPS{ pd}, ca. 42% higher than spring values), and with a 60% reduction in the concentration of total chlorophyll (Chl a+b). These changes were associated with a low capacity of dissipation of the excitation energy non-radiatively (measured as NPQ). Leaves showed a complete recovery of F{ v}/F{ m} and xanthophyll and chlorophyll concentrations after the autumn rainfall, which reached levels similar to that of spring. This poikilohydric-type response of S. tenacissima to stress allows for a greater tolerance of water shortage, high temperature and high light intensity, which are typical in these semi-arid environments and accounts for its distinctive opportunistic growth.

Extensive gene flow blurs phylogeographic but not phylogenetic signal in Olea europaea L.

Genetic structure and evolutionary patterns of the wild olive tree (Olea europaea L.) were investigated with AFLP fingerprinting data at three geographic levels: (a) phylogenetic relationships of the six currently recognized subspecies in Eurasia and Africa; (b) lineage identification in subsp. europaea of the Mediterranean basin; and (c) phylogeography in the western Mediterranean. Two statistical approaches (Bayesian inference and analysis of molecular variance) were used to analyse the AFLP fingerprints. To determine the congruency and transferability of results across studies previous RAPD and ISSR data were analysed in a similar manner. Comparisons proved that qualitative results were mostly congruent but quantitative values differed, depending on the method of analysis. Neighbour-Joining analysis of AFLP phenotypes supported current classification of subspecies. At a Mediterranean scale no clear cut phylogeographic pattern was recovered, likely due to extensive gene flow between populations of subsp. europaea. Gene flow estimates calculated with conventional F-statistics showed that reproductive barriers separated neither populations nor lineages of O. europaea. Genetic divergence between eastern and western parts of the Mediterranean basin was observed only when geographical and population information were incorporated into the analyses through hierarchical analysis of molecular variance (AMOVA). Within the western Mediterranean, the highest genetic diversity was found in two regions: on both sides of the Strait of Gibraltar and in the Balearic archipelago. Additionally, long-lasting isolation of the northern-most populations of the Iberian Peninsula appeared to be responsible for a significant divergence.

Genetic diversity and differentiation processes in the ploidy series of Olea europaea L.: a multiscale approach from subspecies to insular populations

Geographical isolation and polyploidization are central concepts in plant evolution. The hierarchical organization of archipelagos in this study provides a framework for testing the evolutionary consequences for polyploid taxa and populations occurring in isolation. Using amplified fragment length polymorphism and simple sequence repeat markers, we determined the genetic diversity and differentiation patterns at three levels of geographical isolation in Olea europaea: mainland‐archipelagos, islands within an archipelago, and populations within an island. At the subspecies scale, the hexaploid ssp. maroccana (southwest Morocco) exhibited higher genetic diversity than the insular counterparts. In contrast, the tetraploid ssp. cerasiformis (Madeira) displayed values similar to those obtained for the diploid ssp. guanchica (Canary Islands). Geographical isolation was associated with a high genetic differentiation at this scale. In the Canarian archipelago, the stepping‐stone model of differentiation suggested in a previous study was partially supported. Within the western lineage, an east‐to‐west differentiation pattern was confirmed. Conversely, the easternmost populations were more related to the mainland ssp. europaea than to the western guanchica lineage. Genetic diversity across the Canarian archipelago was significantly correlated with the date of the last volcanic activity in the area/island where each population occurs. At the island scale, this pattern was not confirmed in older islands (Tenerife and Madeira), where populations were genetically homogeneous. In contrast, founder effects resulted in low genetic diversity and marked genetic differentiation among populations of the youngest island, La Palma.

Interaction between sulfur dioxide and nitrate in some lichens

Abstract Lichen sensitivity to SO 2 , NO 3 − and to the combination of both pollutants in three fruticose lichen species, Anaptychia ciliaris (L.) Korber, Evernia prunastri (L.) Ach. and Ramalina farinacea (L.) Ach., was studied by evaluating pigment status. A synergistic inhibitory effect of the combination SO 2 + NO 3 − was evident. The role of NO 3 − as a nutrient and as a phytotoxic agent, as well as the differential sensitivity of the species to SO 2 when applied alone, are discussed.

Dark induction of the photoprotective xanthophyll cycle in response to dehydration.

Some plants tolerate tissue dehydration. Dehydration conditions suppress photosynthesis, exacerbating photooxidative stress. In this study, fern samples were collected from the field, desiccated in darkness, and subsequently re-watered. During dark dehydration, zeaxanthin (Z) was formed and maximal photochemical efficiency of PS II was strongly reduced. Rehydration in the dark reversed these effects. Violaxanthin de-epoxidase was responsible for the dark formation of Z as illustrated by its complete inhibition by DTT. Nonetheless, its activity was not affected by nigericin, indicating that Z formation in the dark could be a process independent of the transmembrane pH-gradient into the thylakoids. Synthesis de novo of Z was rejected after blocking carotenogenesis with norfluorazon. Dark formation of Z was also observed in dehydrating leaves of desiccation-intolerant plants, which seems to indicate that this is a phenomenon scattered among different taxa within the plant kingdom. Plants may trigger this mechanism during dehydration, for chlorophyll protection during desiccation, and for faster acclimation when rehydrating conditions return. Violaxanthin de-epoxidation to form Z is typically a light-dependent process, but the formation induced solely by dehydration might represent an anticipatory mechanism for preventing early morning photodamage in desiccation-tolerant plants such as the fern Ceterach officinarum.

Phenotypic plasticity and integration across the canopy of Olea europaea subsp. guanchica (Oleaceae) in populations with different wind exposures.

Woody plants, as sessile and long-lived organisms, are expected to have effective mechanisms for dealing with recurrent environmental stresses. In the present study, we hypothesized that phenotypic plasticity (the ability to express alternative phenotypes) and integration (covariation among functionally related traits) are elicited in plants under stressful wind speed conditions. We investigated the within-crown variation of nine vegetative traits of a tree species (Olea europaea subsp. guanchica) in six populations that represented a gradient of wind speed exposures. Wind-exposed twigs in outer-canopy layers had smaller leaves; thinner, lighter, and shorter internodes; and a larger internode cross-sectional area to leaf area ratio. Comparison between field and greenhouse trials revealed that field differences among populations were mediated by phenotypic plasticity. Outer-canopy twigs expressed plastic responses in populations exposed to high wind speeds, whereas inner-canopy twigs displayed high phenotypic convergence among populations. In addition, phenotypic integration increased with wind exposure (outer canopy > inner canopy > greenhouse) and was consequently affected by canopy openness. We conclude that exposure to wind above a certain speed threshold in this woody species elicits a plastic response that is associated with increased integration among traits and involves mechanical and hydraulic rearrangements in more exposed parts of the trees.

Contrasting patterns of morphological and physiological differentiation across insular environments: phenotypic variation and heritability of light-related traits in Olea europaea.

Phenotypic variation of traits can reflect the ability of plants to adjust to particular environments, but how much of this variation is heritable is not frequently analyzed in natural populations. In the present paper, we investigated the patterns of phenotypic expression in light-related leaf traits of Olea europaea subsp. guanchica, a woody sclerophyllous species endemic to the Canary Islands. We explored phenotypic differentiation and heritable variation across several island populations differing in light environment. A suite of morpho-functional (leaf size, SLA and leaf angle) and physiological (pigment pools: Chl a/b ratio, xantophyll cycle and β-carotene) traits was measured in six populations on three islands. In addition, we estimated heritabilities for these traits following Ritland’s method. Variation in morpho-functional, but not in physiological, traits was observed across the islands and was significantly related to the amount of diffuse light experienced by each population. In addition, significant heritabilities were found for morpho-functional traits, whereas expression of similar phenotypes among populations was accompanied by a lack of heritable variation in physiological traits. Most recently established populations did not exhibit lower heritabilities in quantitative traits than older populations, and apparently displayed congruent phenotypes under the local conditions. Our results strongly support the idea that different types of traits show contrasted levels of genetic and phenotypic variation in populations experiencing marked environmental differences.

Temporal matching among diurnal photosynthetic patterns within the crown of the evergreen sclerophyll Olea europaea L

Trees are modular organisms that adjust their within-crown morphology and physiology in response to within-crown light gradients. However, whether within-plant variation represents a strategy for optimizing light absorption has not been formally tested. We investigated the arrangement of the photosynthetic surface throughout one day and its effects on the photosynthetic process, at the most exposed and most sheltered crown layers of a wild olive tree (Olea europaea L.). Similar measurements were made for cuttings taken from this individual and grown in a greenhouse at contrasted irradiance-levels (100 and 20% full sunlight). Diurnal variations in light interception, carbon fixation and carbohydrate accumulation in sun leaves were negatively correlated with those in shade leaves under field conditions when light intensity was not limiting. Despite genetic identity, these complementary patterns were not found in plants grown in the greenhouse. The temporal disparity among crown positions derived from specialization of the photosynthetic behaviour at different functional and spatial scales: architectural structure (crown level) and carbon budget (leaf level). Our results suggest that the profitability of producing a new module may not only respond to construction costs or light availability, but also rely on its spatio-temporal integration within the productive processes at the whole-crown level.

Long-term responses of the green-algal lichen Parmelia caperata to natural CO2 enrichment

Abstract Acclimation to elevated CO2 was investigated in Parmelia caperata originating from the vicinity of a natural CO2 spring, where the average daytime CO2 concentration was 729 ± 39 μmol mol−1 dry air. Thalli showed no evidence of a down-regulation in photosynthetic capacity following long-term exposure to CO2 enrichment in the field; carboxylation efficiency, total Ribulose bisphosphate carboxylase/oxygenase (Rubisco) content, apparent quantum yield of CO2 assimilation, and the light-saturated rate of CO2 assimilation (measured under ambient and saturating CO2 concentrations) were similar in thalli from the naturally CO2 enriched site and an adjacent control site where the average long-term CO2 concentration was about 355 μmol mol−1. Thalli from both CO2 environments exhibited low CO2 compensation points and early saturation of CO2 uptake kinetics in response to increasing external CO2 concentrations, suggesting the presence of an active carbon-concentrating mechanism. Consistent with the lack of significant effects on photosynthetic metabolism, no changes were found in the nitrogen content of thalli following prolonged exposure to elevated CO2. Detailed intrathalline analysis revealed a decreased investment of nitrogen in Rubisco in the pyrenoid of algae located in the elongation zone of thalli originating from elevated CO2, an effect associated with a reduction in the percentage of the cell volume occupied by lipid bodies and starch grains. Although these differences did not affect the photosynthetic capacity of thalli, there was evidence of enhanced limitations to CO2 assimilation in lichens originating from the CO2-enriched site. The light-saturated rate of CO2 assimilation measured at the average growth CO2 concentration was found to be significantly lower in thalli originating from a CO2-enriched atmosphere compared with that of thalli originating and measured at ambient CO2. At lower photosynthetic photon flux densities, the light compensation point of net CO2 assimilation was significantly higher in thalli originating from elevated CO2, and this effect was associated with higher usnic acid content.