Biological remediation treatments improve the health of a mixed contaminated soil before significantly reducing contaminant levels.

The remediation of mixed contaminated soil is challenging as it often requires actions to minimize metal-induced risks while degrading organic contaminants. Here, the effectiveness of different bioremediation strategies, namely, rhizoremediation with native plant species, mycoremediation with Pleurotus ostreatus spent mushroom substrate, and biostimulation with organic by-products (i.e., composted sewage sludge and spent mushroom substrate), for the recovery of a mixed contaminated soil from an abandoned gravel pit was studied. The combination of biostimulation and rhizoremediation led to the most significant increase in soil health, according to microbial indicator values. The application of composted sewage sludge led to the highest reduction in anthracene and polychlorinated biphenyls concentrations. None of the strategies managed to decrease contamination levels below regulatory limits, but they did enhance soil health. It was concluded that the biological remediation treatments improved soil functioning in a short time, before the concentration of soil contaminants was significantly reduced.

Stress Response to Climate Change and Postharvest Handling in Two Differently Pigmented Lettuce Genotypes: Impact on Alternaria alternata Invasion and Mycotoxin Production.

Many species of Alternaria are important pathogens that cause plant diseases and postharvest rots. They lead to significant economic losses in agriculture and affect human and animal health due to their capacity to produce mycotoxins. Therefore, it is necessary to study the factors that can result in an increase in A. alternata. In this study, we discuss the mechanism by which phenol content protects from A. alternata, since the red oak leaf cultivar (containing higher phenols) showed lower invasion than the green one, Batavia, and no mycotoxin production. A climate change scenario enhanced fungal growth in the most susceptible cultivar, green lettuce, likely because elevated temperature and CO2 levels decrease plant N content, modifying the C/N ratio. Finally, while the abundance of the fungi was maintained at similar levels after keeping the lettuces for four days at 4 °C, this postharvest handling triggered TeA and TEN mycotoxin synthesis, but only in the green cultivar. Therefore, the results demonstrated that invasion and mycotoxin production are cultivar- and temperature-dependent. Further research should be directed to search for resistant cultivars and effective postharvest strategies to reduce the toxicological risk and economic losses related to this fungus, which are expected to increase in a climate change scenario.

Mycorrhizal-Assisted Phytoremediation and Intercropping Strategies Improved the Health of Contaminated Soil in a Peri-Urban Area.

Soils of abandoned and vacant lands in the periphery of cities are frequently subjected to illegal dumping and can undergo degradation processes such as depletion of organic matter and nutrients, reduced biodiversity, and the presence of contaminants, which may exert an intense abiotic stress on biological communities. Mycorrhizal-assisted phytoremediation and intercropping strategies are highly suitable options for remediation of these sites. A two-year field experiment was conducted at a peri-urban site contaminated with petroleum hydrocarbons and polychlorinated biphenyls, to assess the effects of plant growth (spontaneous plant species, Medicago sativa, and Populus × canadensis, alone vs. intercropped) and inoculation of a commercial arbuscular mycorrhizal and ectomycorrhizal inoculum. Contaminant degradation, plant performance, and biodiversity, as well as a variety of microbial indicators of soil health (microbial biomass, activity, and diversity parameters) were determined. The rhizosphere bacterial and fungal microbiomes were assessed by measuring the structural diversity and composition via amplicon sequencing. Establishment of spontaneous vegetation led to greater plant and soil microbial diversity. Intercropping enhanced the activity of soil enzymes involved in nutrient cycling. The mycorrhizal treatment was a key contributor to the establishment of intercropping with poplar and alfalfa. Inoculated and poplar-alfalfa intercropped soils had a higher microbial abundance than soils colonized by spontaneous vegetation. Our study provided evidence of the potential of mycorrhizal-assisted phytoremediation and intercropping strategies to improve soil health in degraded peri-urban areas.

Application of in situ bioremediation strategies in soils amended with sewage sludges.

Increasing soil loss and the scarcity of useful land requires new reusing strategies. Thus, recovery of polluted soils recovery offers a chance for economic and social regeneration. With this objective, different soil cleaning technologies have been developed during the last few decades. On one hand, classical physical and/or chemical technologies can be found which are efficient, but have high costs and impacts upon ecosystems. On the other hand, biological methods (such as phytoremediation, bioremediation and vermiremediation) are relatively cost effective and eco-friendly, but also more time-consuming. These biological methods and their yields have been widely studied but little is known about the interaction between different soil cleaning methods. The combination of different biological strategies could lead to an improvement in remediation performance. Hence, in the present work, different micro-, vermi- and phyto-remediation combinations are applied in a sewage sludge polluted landfill in Gernika-Lumo (Basque Country) which was used as a disposal point for decades, in search of the treatment (single) or combination (dual or triple) of treatments with best remediation yields. Eight experimental groups were applied (n=3) placing earthworms (E), bacteria (B), plants (P), bacteria+earthworms (B+E), bacteria+plants (B+P), plants+earthworms (P+E) plants+bacteria+earthworms (P+B+E) and a non-treated (N.T.) group in the experimental plot (Landfill 17), for 12 months. In order to assess the efficiency of each treatment, a complete characterization (chemical and ecotoxicological) was carried out before and after remediation. Results showed high removal rates for dieldrin (between 50% and 78%) in all the experimental groups. In contrast, removal rates around 20-25% were achieved for heavy metals (Cd 15%-35%; Ni 24%-37%; Pb 15%-33%; Cr 7%-39%) and benzo(a)pyrene (19.5%-28%). The highest reductions were observed in dual (P+E, B+E) and triple (P+B+E) treatments. The best elimination yields were obtained after P+B+E treatment, as highlighted by the battery of ecotoxicological tests and bioassays performed with earthworms, plants and bacteria.

Successful remediation of soils with mixed contamination of chromium and lindane: Integration of biological and physico-chemical strategies.

Soils contaminated by organic and inorganic pollutants like Cr(VI) and lindane, is currently a main environmental challenge. Biological strategies, such as biostimulation, bioaugmentation, phytoremediation and vermiremediation, and nanoremediation with nanoscale zero-valent iron (nZVI) are promising approaches for polluted soil health recovery. The combination of different remediation strategies might be key to address this problem. For this reason, a greenhouse experiment was performed using soil without or with an organic amendment. Both soils were contaminated with lindane (15 mg kg-1) and Cr(VI) (100 or 300 mg kg-1). After one month of aging, the following treatments were applied: (i) combination of bioaugmentation (actinobacteria), phytoremediation (Brassica napus), and vermiremediation (Eisenia fetida), or (ii) nanoremediation with nZVI, or (iii) combination of biological treatments and nanoremediation. After 60 days, the wellness of plants and earthworms was assessed, also, soil health was evaluated through physico-chemical parameters and biological indicators. Cr(VI) was more toxic and decreased soil health, however, it was reduced to Cr(III) by the amendment and nZVI and, to a lesser extent, by the biological treatment. Lindane was more effectively degraded through bioremediation. In non-polluted soils, nZVI had strong deleterious effects on soil biota when combined with the organic matter, but this effect was reverted in soils with a high concentration of Cr(VI). Therefore, under our experimental conditions bioremediation might be the best for soils with a moderate concentration of Cr(VI) and organic matter. The application of nZVI in soils with a high content of organic matter should be avoided except for soils with very high concentrations of Cr(VI). According to our study, among the treatments tested, the combination of an organic amendment, biological treatment, and nZVI was shown to be the strategy of choice in soils with high concentrations of Cr(VI) and lindane, while for moderate levels of chromium, the organic amendment plus biological treatment is the most profitable treatment.

Gentle remediation options for soil with mixed chromium (VI) and lindane pollution: biostimulation, bioaugmentation, phytoremediation and vermiremediation.

Gentle Remediation Options (GROs), such as biostimulation, bioaugmentation, phytoremediation and vermiremediation, are cost-effective and environmentally-friendly solutions for soils simultaneously polluted with organic and inorganic compounds. This study assessed the individual and combined effectiveness of GROs in recovering the health of a soil artificially polluted with hexavalent chromium [Cr(VI)] and lindane. A greenhouse experiment was performed using organically-amended vs. non-amended mixed polluted soils. All soils received the following treatments: (i) no treatment; (ii) bioaugmentation with an actinobacteria consortium; (iii) vermiremediation with Eisenia fetida; (iv) phytoremediation with Brassica napus; (v) bioaugmentation + vermiremediation; (vi) bioaugmentation + phytoremediation; and (vii) bioaugmentation + vermiremediation + phytoremediation. Soil health recovery was determined based on Cr(VI) and lindane concentrations, microbial properties and toxicity bioassays with plants and worms. Cr(VI) pollution caused high toxicity, but some GROs were able to partly recover soil health: (i) the organic amendment decreased Cr(VI) concentrations, alleviating toxicity; (ii) the actinobacteria consortium was effective at removing both Cr(VI) and lindane; (iii) B. napus and E. fetida had a positive effect on the removal of pollutants and improved microbial properties. The combination of the organic amendment, B. napus, E. fetida and the actinobacteria consortium was the most effective strategy.

Born to revive: molecular and physiological mechanisms of double tolerance in a paleotropical and resurrection plant.

Resurrection plants recover physiological functions after complete desiccation. Almost all of them are native to tropical warm environments. However, the Gesneriaceae include four genera, remnant of the past palaeotropical flora, which inhabit temperate mountains. One of these species is additionally freezing-tolerant: Ramonda myconi. We hypothesise that this species has been able to persist in a colder climate thanks to some resurrection-linked traits. To disentangle the physiological mechanisms underpinning multistress tolerance to desiccation and freezing, we conducted an exhaustive seasonal assessment of photosynthesis (gas exchange, limitations to partitioning, photochemistry and galactolipids) and primary metabolism (through metabolomics) in two natural populations at different elevations. R. myconi displayed low rates of photosynthesis, largely due to mesophyll limitation. However, plants were photosynthetically active throughout the year, excluding a reversible desiccation period. Common responses to desiccation and low temperature involved chloroplast protection: enhanced thermal energy dissipation, higher carotenoid to Chl ratio and de-epoxidation of the xanthophyll cycle. As specific responses, antioxidants and secondary metabolic routes rose upon desiccation, while putrescine, proline and a variety of sugars rose in winter. The data suggest conserved mechanisms to cope with photo-oxidation during desiccation and cold events, while additional metabolic mechanisms may have evolved as specific adaptations to cold during recent glaciations.

A field portable method for the semi-quantitative estimation of dehydration tolerance of photosynthetic tissues across distantly related land plants.

Desiccation tolerant (DT) plants withstand complete cellular dehydration, reaching relative water contents (RWC) below 30% in their photosynthetic tissues. Desiccation sensitive (DS) plants exhibit different degrees of dehydration tolerance (DHT), never surviving water loss >70%. To date, no procedure for the quantitative evaluation of DHT extent exists that is able to discriminate DS species with differing degrees of DHT from truly DT plants. We developed a simple, feasible and portable protocol to differentiate between DT and different degrees of DHT in the photosynthetic tissues of seed plants and between fast desiccation (< 24 h) tolerant (FDT) and sensitive (FDS) bryophytes. The protocol is based on (1) controlled desiccation inside Falcon tubes equilibrated at three different relative humidities that, consequently, induce three different speeds and extents of dehydration and (2) an evaluation of the average percentage of maximal photochemical efficiency of PSII (Fv /fm) recovery after rehydration. Applying the method to 10 bryophytes and 28 tracheophytes from various locations, we found that (1) imbibition of absorbent material with concentrated salt-solutions inside the tubes provides stable relative humidity and avoids direct contact with samples; (2) for 50 ml capacity tubes, the optimal plant amount is 50-200 mg fresh weight; (3) the method is useful in remote locations due to minimal instrumental requirements; and (4) a threshold of 30% recovery of the initial Fv /fm upon reaching RWC ≤ 30% correctly categorises DT species, with three exceptions: two poikilochlorophyllous species and one gymnosperm. The protocol provides a semi-quantitative expression of DHT that facilitates comparisons of species with different morpho-physiological traits and/or ecological attributes.

Can Parietin Transfer Energy Radiatively to Photosynthetic Pigments?

The main role of lichen anthraquinones is in protection against biotic and abiotic stresses, such as UV radiation. These compounds are frequently deposited as crystals outside the fungal hyphae and most of them emit visible fluorescence when excited by UV. We wondered whether the conversion of UV into visible fluorescence might be photosynthetically used by the photobiont, thereby converting UV into useful energy. To address this question, thalli of Xanthoria parietina were used as a model system. In this species the anthraquinone parietin accumulates in the outer upper cortex, conferring the species its characteristic yellow-orange colouration. In ethanol, parietin absorbed strongly in the blue and UV-B and emitted fluorescence in the range 480⁻540 nm, which partially matches with the absorption spectra of photosynthetic pigments. In intact thalli, it was determined by confocal microscopy that fluorescence emission spectra shifted 90 nm towards longer wavelengths. Then, to study energy transfer from parietin, we compared the response to UV of untreated and parietin-free thalli (removed with acetone). A chlorophyll fluorescence kinetic assessment provided evidence of UV-induced electron transport, though independently of the presence of parietin. Thus, a role for anthraquinones in energy harvesting is not supported for X. parietina under presented experimental conditions.

Effectiveness and ecotoxicity of zero-valent iron nanoparticles during rhizoremediation of soil contaminated with Zn, Cu, Cd and diesel.

The remediation of soils simultaneously contaminated with organic and inorganic compounds is still a challenging task. The application of metallic nanoparticles, such as zero-valent iron nanoparticles (nZVI), for soil remediation is highly promising, but their effectiveness and potential ecotoxicity must be further investigated. In addition, the performance of nZVI when combined with other remediation strategies is a topic of great interest. Here, we present data on soil chemical (pseudo-total and CaCl2-extractable metal concentrations; petroleum hydrocarbon concentrations) and biological properties (microbial properties and phytotoxicity) after the application of nZVI to soil simultaneously contaminated with Zn, Cu, Cd and diesel, in the absence and presence of other remediation treatments such as the application of an organic amendment and the growth of Brassica napus plants. Soils were artificially contaminated with the abovementioned contaminants. Then, after an aging period of one month, nZVI were applied to the soil and, subsequently, B. napus seeds were sown. Plants were left to grow for one month. Soil samples were collected immediately after artificially contaminating the soil (T1), at sowing (T2) and at harvesting (T3). Overall, the application of nZVI had no effect on contaminant removal, nor on soil microbial parameters. In contrast, it did cause an indirect toxic effect on plant root elongation due to the interaction of nZVI with soil organic matter. These data are useful for researchers and companies interested in the effectiveness and ecotoxicity of zero-valent iron nanoparticles during the remediation of soil contaminated with metals and hydrocarbons, especially when combined with Gentle Remediation Options.

Brassica napus has a key role in the recovery of the health of soils contaminated with metals and diesel by rhizoremediation.

Contaminated soils are frequently characterized by the simultaneous presence of organic and inorganic contaminants, as well as a poor biological and nutritional status. Rhizoremediation, the combined use of phytoremediation and bioremediation, has been proposed as a Gentle Remediation Option to rehabilitate multi-contaminated soils. Recently, newer techniques, such as the application of metallic nanoparticles, are being deployed in an attempt to improve traditional remediation options. In order to implement a phytomanagement strategy on calcareous alkaline peri-urban soils simultaneously contaminated with several metals and diesel, we evaluated the effectiveness of Brassica napus L., a profitable crop species, assisted with organic amendment and zero-valent iron nanoparticles (nZVI). A two-month phytotron experiment was carried out using two soils, i.e. amended and unamended with organic matter. Soils were artificially contaminated with Zn, Cu and Cd (1500, 500 and 50mgkg-1, respectively) and diesel (6000mgkg-1). After one month of stabilization, soils were treated with nZVI and/or planted with B. napus. The experiment was conducted with 16 treatments resulting from the combination of the following factors: amended/unamended, contaminated/non-contaminated, planted/unplanted and nZVI/no-nZVI. Soil physicochemical characteristics and biological indicators (plant performance and soil microbial properties) were determined at several time points along the experiment. Carbonate content of soils was the crucial factor for metal immobilization and, concomitantly, reduction of metal toxicity. Organic amendment was essential to promote diesel degradation and to improve the health and biomass of B. napus. Soil microorganisms degraded preferably diesel hydrocarbons of biological origin (biodiesel). Plants had a remarkable positive impact on the activity and functional diversity of soil microbial communities. The nZVI were ineffective as soil remediation tools, but did not cause any toxicity. We concluded that rhizoremediation with B. napus combined with an organic amendment is promising for the phytomanagement of calcareous soils with mixed (metals and diesel) contamination.

Photoprotective Strategies of Mediterranean Plants in Relation to Morphological Traits and Natural Environmental Pressure: A Meta-Analytical Approach.

Despite being a small geographic extension, Mediterranean Basin is characterized by an exceptional plant biodiversity. Adaptive responses of this biocoenosis are delineated by an unusual temporal dissociation along the year between optimal temperature for growth and water availability. This fact generates the combination of two environmental stress factors: a period of summer drought, variable in length and intensity, and the occurrence of mild to cold winters. Both abiotic factors, trigger the generation of (photo)oxidative stress and plants orchestrate an arsenal of structural, physiological, biochemical, and molecular mechanisms to withstand such environmental injuries. In the last two decades an important effort has been made to characterize the adaptive morphological and ecophysiological traits behind plant survival strategies with an eye to predict how they will respond to future climatic changes. In the present work, we have compiled data from 89 studies following a meta-analytical approach with the aim of assessing the composition and plasticity of photosynthetic pigments and low-molecular-weight antioxidants (tocopherols, glutathione, and ascorbic acid) of wild Mediterranean plant species. The influence of internal plant and leaf factors on such composition together with the stress responsiveness, were also analyzed. This approach enabled to obtain data from 73 species of the Mediterranean flora, with the genus Quercus being the most frequently studied. Main highlights of present analysis are: (i) sort of photoprotective mechanisms do not differ between Mediterranean plants and other floras but they show higher plasticity indexes; (ii) α-tocopherol among the antioxidants and violaxanthin-cycle pigments show the highest responsiveness to environmental factors; (iii) both winter and drought stresses induce overnight retention of de-epoxidised violaxanthin-cycle pigments; (iv) this retention correlates with depressions of Fv/Fm; and (v) contrary to what could be expected, mature leaves showed higher accumulation of hydrophilic antioxidants than young leaves, and sclerophyllous leaves higher biochemical photoprotective demand than membranous leaves. In a global climatic change scenario, the plasticity of their photoprotective mechanisms will likely benefit Mediterranean species against oceanic ones. Nevertheless, deep research of ecoregions other than the Mediterranean Basin will be needed to fully understand photoprotection strategies of this extremely biodiverse floristic biome: the Mediterranean ecosystem.

Multi-targeted metagenetic analysis of the influence of climate and environmental parameters on soil microbial communities along an elevational gradient.

Mountain elevation gradients are invaluable sites for understanding the effects of climate change on ecosystem function, community structure and distribution. However, relatively little is known about the impact on soil microbial communities, in spite of their importance for the functioning of the soil ecosystem. Previous studies of microbial diversity along elevational gradients were often limited by confounding variables such as vegetation, pH, and nutrients. Here, we utilised a transect in the Pyrenees established to minimise variation in such parameters, to examine prokaryotic, fungal, protist and metazoan communities throughout three consecutive years. We aimed to determine the influences of climate and environmental parameters on soil microbial community structure; as well as on the relationships between those microbial communities. Further, functional diversity of heterotrophic bacteria was determined using Biolog. Prokaryotic and fungal community structure, but not alpha-diversity, correlated significantly with elevation. However, carbon-to-nitrogen ratio and pH appeared to affect prokaryotic and protist communities more strongly. Both community structure and physicochemical parameters varied considerably between years, illustrating the value of long-term monitoring of the dynamic processes controlling the soil ecosystem. Our study also illustrates both the challenges and strengths of using microbial communities as indicators of potential impacts of climate change.

Enhancement of ecosystem services during endophyte-assisted aided phytostabilization of metal contaminated mine soil.

Endophytic plant growth-promoting bacteria (endophytes) were isolated from a variety of (pseudo)metallophytes growing in an abandoned Zn/Pb mine and then characterized according to their plant growth-promoting traits (i.e. ACC deaminase activity, IAA production, siderophore production, phosphate solubilising capacity, metal and salt tolerance and phenotypic characterization). Initially, under growth chamber conditions, an endophyte-assisted aided phytostabilization study was carried out with Festuca rubra plants (native vs. commercial variety) inoculated with a Pseudomonas sp. isolate and cow slurry as organic amendment. The effect of treatments on soil physicochemical and microbial indicators of soil quality, as well as plant physiological parameters and metal concentrations, was assessed. We performed a complementary interpretation of our data through their grouping within a set of ecosystem services. Although the application of cow slurry had the most pronounced effects on soil quality indicators and ecosystem services, the growth of native F. rubra plants reduced soil bioavailability of Cd and Zn by 19 and 22%, respectively, and enhanced several soil microbial parameters. On the other hand, endophyte (Pseudomonas sp.) inoculation improved the physiological status of F. rubra plants by increasing the content of carotenoids, chlorophylls and Fv/Fm by 69, 65 and 37%, respectively, while also increasing the values of several soil microbial parameters. Finally, a consortium of five endophyte isolates was used for an endophyte-assisted aided phytostabilization field experiment, where lower metal concentrations in native excluder plants were found. Nonetheless, the field inoculation of the endophyte consortium had no effect on the biomass of native plants.

Ecophysiological roles of abaxial anthocyanins in a perennial understorey herb from temperate deciduous forests.

Accumulation of abaxial anthocyanins is an intriguing leaf trait particularly common among deeply shaded understorey plants of tropical and temperate forests whose ecological significance is still not properly understood. To shed light on it, possible ecophysiological roles of abaxial anthocyanins were tested in the perennial understorey herb of temperate deciduous forests Saxifraga hirsuta, chosen as a model species due to the coexistence of green and anthocyanic leaves and the presence of an easily removable lower anthocyanic epidermis. Anthocyanins accumulated during autumn, which temporally matched the overstorey leaf fall. Patterns of development of abaxial anthocyanins and direct measurements of photochemical efficiency under monochromatic light were not consistent with a photoprotective hypothesis. Enhancement of light capture also seemed unlikely since the back-scattering of red light towards the lower mesophyll was negligible. Seed germination was similar under acyanic and anthocyanic leaves. A relevant consequence of abaxial anthocyanins was the dramatic reduction of light transmission through the leaf. The dark environment generated underneath the Saxifraga canopy was enhanced by the horizontal repositioning of leaves, which occurs in parallel with reddening. This might play a role in biotic interactions by inhibiting vital processes of competitors, which may be of especial importance in spring before the overstorey leaves sprout.

Internal and external factors affecting photosynthetic pigment composition in plants: a meta-analytical approach.

Photosynthetic pigment composition has been a major study target in plant ecophysiology during the last three decades. Although more than 2000 papers have been published, a comprehensive evaluation of the responses of photosynthetic pigment composition to environmental conditions is not yet available. After an extensive survey, we compiled data from 525 papers including 809 species (subkingdom Viridiplantae) in which pigment composition was described. A meta-analysis was then conducted to assess the ranges of photosynthetic pigment content. Calculated frequency distributions of pigments were compared with those expected from the theoretical pigment composition. Responses to environmental factors were also analysed. The results revealed that lutein and xanthophyll cycle pigments (VAZ) were highly responsive to the environment, emphasizing the high phenotypic plasticity of VAZ, whereas neoxanthin was very stable. The present meta-analysis supports the existence of relatively narrow limits for pigment ratios and also supports the presence of a pool of free 'unbound' VAZ. Results from this study provide highly reliable ranges of photosynthetic pigment contents as a framework for future research on plant pigments.

Evidence for the absence of enzymatic reactions in the glassy state. A case study of xanthophyll cycle pigments in the desiccation-tolerant moss Syntrichia ruralis.

Desiccation-tolerant plants are able to withstand dehydration and resume normal metabolic functions upon rehydration. These plants can be dehydrated until their cytoplasm enters a 'glassy state' in which molecular mobility is severely reduced. In desiccation-tolerant seeds, longevity can be enhanced by drying and lowering storage temperature. In these conditions, they still deteriorate slowly, but it is not known if deteriorative processes include enzyme activity. The storage stability of photosynthetic organisms is less studied, and no reports are available on the glassy state in photosynthetic tissues. Here, the desiccation-tolerant moss Syntrichia ruralis was dehydrated at either 75% or <5% relative humidity, resulting in slow (SD) or rapid desiccation (RD), respectively, and different residual water content of the desiccated tissues. The molecular mobility within dry mosses was assessed through dynamic mechanical thermal analysis, showing that at room temperature only rapidly desiccated samples entered the glassy state, whereas slowly desiccated samples were in a 'rubbery' state. Violaxanthin cycle activity, accumulation of plastoglobules, and reorganization of thylakoids were observed upon SD, but not upon RD. Violaxanthin cycle activity critically depends on the activity of violaxanthin de-epoxidase (VDE). Hence, it is proposed that enzymatic activity occurred in the rubbery state (after SD), and that in the glassy state (after RD) no VDE activity was possible. Furthermore, evidence is provided that zeaxanthin has some role in recovery apparently independent of its role in non-photochemical quenching of chlorophyll fluorescence.

Patterns of spatio-temporal distribution of winter chronic photoinhibition in leaves of three evergreen Mediterranean species with contrasting acclimation responses.

High irradiance and relatively low temperature, which characterize Mediterranean winters, cause chilling stress in plants. Downregulation of photosynthetic efficiency is a mechanism that allows plants to survive these conditions. This study aims to address whether this process shows a regular spatial pattern across leaf surface or not. Three species (Buxus sempervirens, Cistus albidus and Arctostaphylos uva-ursi) with contrasting responses to winter stress were studied. During 7 days, macro and micro Fv/Fm spatial patterns were monitored by the use of chlorophyll fluorescence imaging techniques. In the field, the strongest photoinhibition was found in B. sempervirens, while there was almost no chronic photoinhibition in C. albidus. In leaves of the first species, Fv/Fm decreased from base to tip while in C. albidus it was uniform over the leaf lamina. An intermediate behavior is shown by A. uva-ursi leaves. Spatial heterogeneity distribution of Fv/Fm was found inside the leaves, resulting in greater Fv/Fm values in the inner layers than in the outer ones. Neither xanthophyll-linked downregulation of Fv/Fm nor protein remobilization were the reasons for such spatial patterns since pigment composition and nitrogen content did not reveal tip-base differences. During recovery from winter, photoinhibition changes occurred in Fv/Fm, pigments and chloroplast ultrastructure. This work shows for the first time that irrespective of physiological mechanisms responsible for development of winter photoinhibition, there is an acclimation response with strong spatio-temporal variability at leaf level in some species. This observation should be taken into account when modeling or scaling up photosynthetic responses.

Alternative methods for sampling and preservation of photosynthetic pigments and tocopherols in plant material from remote locations.

Current methods for the study of pigments involve freezing in liquid nitrogen and storage at -80 degrees C or lyophilization until HPLC analysis. These requirements greatly restrict ecophysiological research in remote areas where such resources are hardly available. We aimed to overcome such limitations by developing several techniques not requiring freezing or lyophilization. Two species with contrasting foliar characteristics (Olea europaea and Taraxacum officinale) were chosen. Seven preservation methods were designed, optimized and tested in a field trial. These protocols were compared with a control immediately frozen after collection. Pigments and tocopherols were analysed by HPLC. Main artefacts were chlorophyll epimerization or phaeophytinization, carotenoid isomerization, altered de-epoxidation index and tocopherol degradation. Among all methods, sample desiccation in silica gel provides robust samples (pigment composition was unaffected by storage time or temperature) and almost unaltered pigment profiles, except for a shift in epoxidation state. Although liquid nitrogen freezing and subsequent lyophilization or freezer storage were preferred, when these facilities are either not available or not suitable for long-distance transport, desiccation with silica gel, passive extraction in acetone and/or storage of fresh samples in water vapour saturated atmospheres enable a complete pigment characterization. Silica gel is advisable for long-term sample conservation.