The Tripartite Lichen Ricasolia virens: Involvement of Cyanobacteria and Bacteria in Its Morphogenesis.

Ricasolia virens is an epiphytic lichen-forming fungus mainly distributed in Western Europe and Macaronesia in well-structured forests with ecological continuity that lack eutrophication. It is considered to be threatened or extinct in many territories in Europe (IUCN). Despite its biological and ecological relevance, studies on this taxon are scarce. The thalli are tripartite, and the mycobiont has a simultaneous symbiotic relationship with cyanobacteria and green microalgae, which represent interesting models to analyse the strategies and adaptations resulting from the interactions of lichen symbionts. The present study was designed to contribute to a better understanding of this taxon, which has shown a clear decline over the last century. The symbionts were identified by molecular analysis. The phycobiont is Symbiochloris reticulata, and the cyanobionts (Nostoc) are embedded in internal cephalodia. Light, transmission electron and low-temperature scanning microscopy techniques were used to investigate the thallus anatomy, ultrastructure of microalgae and ontogeny of pycnidia and cephalodia. The thalli are very similar to its closest relative, Ricasolia quercizans. The cellular ultrastructure of S. reticulata by TEM is provided. Non-photosynthetic bacteria located outside the upper cortex are introduced through migratory channels into the subcortical zone by the splitting of fungal hyphae. Cephalodia were very abundant, but never as external photosymbiodemes.

Effect of sulfur on pollen germination of Clemenules mandarin and Nova tangelo.

This study aims to elucidate whether sulfur can inhibit citrus pollination by affecting pollen grains. For this, four sulfur-based products (inorganic sulfur, water dispersible granular sulfur, ammonium sulfate, copper sulfate) were tested to evaluate their effect on pollen germination and pollen tube growth of two citrus varieties: Clemenules mandarin (Citrus clementina) and Nova tangelo (Citrus clementina x [Citrus paradisi x Citrus reticulata]). Pollen grains were extracted from the flowers of these two varieties and subsequently placed in Petri dishes with modified BK (boron and potassium) germination medium with six concentrations of the sulfur-based products (0.2, 2, 20, 200, 2,000, 20,000 mg l-1). All the dishes were incubated and the pollen germination rate was calculated. All the sulfur products showed progressive pollen germination inhibition with a rising sulfur concentration. CTC50 (50% cytotoxicity inhibition) was around 20 mg l-1, with significant differences among treatments. Total pollen germination inhibition took place at 20,000 mg l-1. These results demonstrate that sulfur application can affect citrus pollination.

Effect of temperature on pollen germination for several Rosaceae species: influence of freezing conservation time on germination patterns.

Between February 2018 and April 2018, flowers were collected from eight Rosaceae species. Flowers were kept in a freezer at -20 °C for three freezing times (Treatment 1, two months; Treatment 2, four months; Treatment 3, six months). After extracting pollen, in vitro germination was induced in a culture medium and incubated at six different temperatures for 72 h. The percentage of pollen germination, average pollen tube length and maximum pollen tube length were measured. Pollen germination was maximum for all species between 15 °C and 30 °C. Cydonia oblonga, Malus sylvestris, Prunus avium, Prunus domestica, Prunus dulcis, Prunus persica and Pyrus communis obtained 30-52% pollen germination between 15 °C and 20 °C. Prunus cerasifera had 40% pollen germination at 30 °C. All species studied reached the maximum pollen tube length between 10 °C and 25 °C. Germination did not change significantly for any of the species with freezing time, but we found significant differences in the three parameters measured between treatments. The highest germination percentages were obtained in Treatment 2 (four months frozen at -20 °C), while the maximum pollen tube length was reached in Treatment 1 (two months frozen at -20 °C). According to our results, freezing time affected the germination-temperature patterns. This could indicate that studies on the effect of temperature on pollen germination should always be carried out with fresh pollen to obtain more conclusive data.

Inhibition of NO Biosynthetic Activities during Rehydration of Ramalina farinacea Lichen Thalli Provokes Increases in Lipid Peroxidation.

Lichens are poikilohydrous symbiotic associations between a fungus, photosynthetic partners, and bacteria. They are tolerant to repeated desiccation/rehydration cycles and adapted to anhydrobiosis. Nitric oxide (NO) is a keystone for stress tolerance of lichens; during lichen rehydration, NO limits free radicals and lipid peroxidation but no data on the mechanisms of its synthesis exist. The aim of this work is to characterize the synthesis of NO in the lichen Ramalina farinacea using inhibitors of nitrate reductase (NR) and nitric oxide synthase (NOS), tungstate, and NG-nitro-L-arginine methyl ester (L-NAME), respectively. Tungstate suppressed the NO level in the lichen and caused an increase in malondialdehyde during rehydration in the hyphae of cortex and in phycobionts, suggesting that a plant-like NR is involved in the NO production. Specific activity of NR in R. farinacea was 91 µU/mg protein, a level comparable to those in the bryophyte Physcomitrella patens and Arabidopsis thaliana. L-NAME treatment did not suppress the NO level in the lichens. On the other hand, NADPH-diaphorase activity cytochemistry showed a possible presence of a NOS-like activity in the microalgae where it is associated with cytoplasmatic vesicles. These data provide initial evidence that NO synthesis in R. farinacea involves NR.

Characterization of the responses to saline stress in the symbiotic green microalga Trebouxia sp. TR9.

MAIN CONCLUSION: For the first time we provide a study on the physiological, ultrastructural and molecular effects of salt stress on a terrestrial symbiotic green microalga, Trebouxia sp. TR9. Although tolerance to saline conditions has been thoroughly studied in plants and, to an extent, free-living microalgae, scientific data regarding salt stress on symbiotic lichen microalgae is scarce to non-existent. Since lichen phycobionts are capable of enduring harsh, restrictive and rapidly changing environments, it is interesting to study the metabolic machinery operating under these extreme conditions. We aim to determine the effects of prolonged exposure to high salt concentrations on the symbiotic phycobiont Trebouxia sp. TR9, isolated from the lichen Ramalina farinacea. Our results suggest that, when this alga is confronted with extreme saline conditions, the cellular structures are affected to an extent, with limited chlorophyll content loss and photosynthetic activity remaining after 72 h of exposure to 5 M NaCl. Furthermore, this organism displays a rather different molecular response compared to land plants and free-living halophile microalgae, with no noticeable increase in ABA levels and ABA-related gene expression until the external NaCl concentration is raised to 3 M NaCl. Despite this, the ABA transduction pathway seems functional, since the ABA-related genes tested are responsive to exogenous ABA. These observations could suggest that this symbiotic green alga may have developed alternative molecular pathways to cope with highly saline environments.

Molecular and morphological diversity of Trebouxia microalgae in sphaerothallioid Circinaria spp. lichens1.

Three vagrant (Circinaria hispida, Circinaria gyrosa, and Circinaria sp. 'paramerae') and one crustose (semi-vagrant, Circinaria sp. 'oromediterranea') lichens growing in very continental areas in the Iberian Peninsula were selected to study the phycobiont diversity. Mycobiont identification was checked using nrITS DNA barcoding: Circinaria sp. 'oromediterranea' and Circinaria sp. 'paramerae' formed a new clade. Phycobiont diversity was analyzed in 50 thalli of Circinaria spp. using nrITS DNA and LSU rDNA, with microalgae coexistence being found in all the species analyzed by Sanger sequencing. The survey of phycobiont diversity showed up to four different Trebouxia spp. as the primary phycobiont in 20 thalli of C. hispida, in comparison with the remaining Circinaria spp., where only one Trebouxia was the primary microalga. In lichen species showing coexistence, some complementary approaches are needed (454 pyrosequencing and/or ultrastructural analyses). Five specimens were selected for high-throughput screening (HTS) analyses: 22 Trebouxia OTUs were detected, 10 of them not previously known. TEM analyses showed three different cell morphotypes (Trebouxia sp. OTU A12, OTU S51, and T. cretacea) whose ultrastructure is described here in detail for the first time. HTS revealed a different microalgae pool in each species studied, and we cannot assume a specific pattern between these pools and the ecological and/or morphological characteristics. The mechanisms involved in the selection of the primary phycobiont and the other microalgae by the mycobiont are unknown, and require complex experimental designs. The systematics of the genus Circinaria is not yet well resolved, and more analyses are needed to establish a precise delimitation of the species.

Coordinated ultrastructural and phylogenomic analyses shed light on the hidden phycobiont diversity of Trebouxia microalgae in Ramalina fraxinea.

The precise boundary delineations between taxa in symbiotic associations are very important for evolutionary and ecophysiological studies. Growing evidence indicates that in many cases, the use of either morphological characters or molecular markers results in diversity underestimation. In lichen symbioses, Trebouxia is the most common genus of lichen phycobionts, however, the diversity within this genus has been poorly studied and as such there is no clear species concept. This study constitutes a multifaceted approach incorporating aspects of ultrastructural characterization by TEM and phylogenomics to evaluate the morphological and genetic diversity of phycobionts within the sexually reproducing lichen Ramalina fraxinea in the context of Mediterranean and temperate populations. Results reveal an association with at least seven different Trebouxia lineages belonging to at least two species, T. decolorans and T. jamesii, and diverse combinations of such lineages coexisting within the same thallus depending on the analyzed sample. Some of these lineages are shared by several other non-related lichen taxa. Our findings indicate the existence of a highly diverse assemblage of Trebouxia algae associating with R. fraxinea and suggest a possible incipient speciation within T. decolorans rendering a number of lineages or even actual species. This study stresses the importance of coordinated ultrastructural and molecular analyses to improve estimates of diversity and reveal the coexistence of more than one Trebouxia species within the same thallus. It is also necessary to have clearer species delimitation criteria within the genus Trebouxia and microalgae in general.

Molecular phylogeny and ultrastructure of the lichen microalga Asterochloris mediterranea sp. nov. from Mediterranean and Canary Islands ecosystems.

The microalgae of the genus Asterochloris are the preferential phycobionts in Cladonia, Lepraria and Stereocaulon lichens. Recent studies have highlighted the hidden diversity of the genus, even though phycobionts hosting species of the genus Cladonia in Mediterranean and Canarian ecosystems have been poorly explored. Phylogenetic analyses were made by concatenation of the sequences obtained with a plastid - LSU rDNA - and two nuclear - internal transcribed spacer (ITS) rDNA and actin - molecular markers of the phycobionts living in several populations of the Cladonia convoluta-Cladonia foliacea complex, Cladonia rangiformis and Cladonia cervicornis s. str. widely distributed in these areas in a great variety of substrata and habitats. A new strongly supported clade was obtained in relation to the previously published Asterochloris phylogenies. Minimum genetic variation was detected between our haplotypes and other sequences available in the GenBank database. The correct identification of the fungal partners was corroborated by the ITS rDNA barcode. In this study we provide a detailed characterization comprising chloroplast morphology, and ultrastructural and phylogenetic analyses of a novel phycobiont species, here described as Asterochloris mediterranea sp. nov. Barreno, Chiva, Moya et Skaloud. A cryopreserved holotype specimen has been deposited in the Culture Collection of Algae of Charles University in Prague, Czech Republic (CAUP) as CAUP H 1015. We suggest the use of a combination of several nuclear and plastid molecular markers, as well as ultrastructural (transmission electron and confocal microscopy) techniques, both in culture and in the symbiotic state, to improve novel species delimitation of phycobionts in lichens.

Different strategies to achieve Pb-tolerance by the two Trebouxia algae coexisting in the lichen Ramalina farinacea.

Lichen thalli are permeable to airborne substances, including heavy metals, which are harmful to cell metabolism. Ramalina farinacea shows a moderate tolerance to Pb. This lichen comprises two Trebouxia phycobionts, provisionally referred to as TR1 and TR9, with distinct physiological responses to acute oxidative stress. Thus, there is a more severe decay in photosynthesis and photosynthetic pigments in TR1 than in TR9. Similarly, under oxidative stress, antioxidant enzymes and HSP70 protein decrease in TR1 but increase in TR9. Since Pb toxicity is associated with increased ROS formation, we hypothesized greater Pb tolerance in this phycobiont. Accordingly, the aim of the present study was to characterize the physiological differences in the responses of TR1 and TR9 to Pb exposure. Liquid cultures of isolated phycobionts were incubated for 7 days in the presence of Pb(NO3)2. Thereafter, extracellular and intracellular Pb accumulation, photosynthetic pigments, and photosynthesis (as modulated chlorophyll fluorescence) were analyzed along with the antioxidant enzymes glutathione reductase (GR), superoxide dismutase (SOD), ascorbate peroxidase (APx), and catalase (CAT), and the stress-related protein HSP70. Pb uptake increased with the amount of supplied Pb in both algae. However, while significantly more metal was immobilized extracellularly by TR9, the amount of intracellular Pb accumulation was three times higher in TR1. In neither of the phycobionts were significant effects on photosynthetic pigments or photosynthetic electron transport observed. While under control conditions GR, SOD, and APx levels were significantly higher in TR1 than in TR9, only in the latter were these enzymes induced by Pb. This resulted in quantitatively similar antioxidant activities in the two algae when exposed to Pb. In conclusion, the phycobionts of R. farinacea make use of two different strategies against stress, in which the integration of distinct anatomical and physiological features affords similar levels of Pb tolerance.

Ultrastructural and histochemical analysis reveals ethylene-induced responses underlying reduced peel collapse in detached citrus fruit.

Fruits from many citrus cultivars develop depressed areas in the flavedo (outer part of the peel) and albedo (inner part) following detachment. Although ultrastructural analysis may provide important information about multiple plant responses to stresses and external stimuli at the cell and tissue levels, and despite the proved efficacy of ethylene in reducing peel damage in citrus fruit, cytological responses of this horticultural crop to protective ethylene concentrations have not yet been reported. We show that applying high ethylene levels (2 µL L(-1) for 14 days) causes sublethal stress as it favored the alteration of cuticle, vacuole, middle lamella and primary wall, especially in the albedo cells, but reduced peel collapse in detached mature "Navelate" oranges (C. sinensis, L. Osbeck) held under nonstressful environmental conditions (22°C and 90-95% RH). Ethylene did not induce relevant changes in lignification but favored the deposition of pectic exudates and the release of sugars from degradation of cell polysaccharides including starch, cellulose, and pectins. In contrast, inhibiting ethylene perception by applying 1-methylcyclopropene (1-MCP) reduced these ethylene-related responses and favored degradation of cell membranes and peel damage. The overall results reflect that mature oranges tolerate high ethylene levels that might favor the activation of defense responses involving oxidative-stress related mechanisms and recycling of nutrients and carbon supply to enable cells to sustain respiration and cope with carbon deprivation stress caused by detachment.

Physiological, anatomical and biomass partitioning responses to ozone in the Mediterranean endemic plant Lamottea dianae.

Ozone effects on the perennial forb Lamottea dianae were studied in an open-top chamber experiment. Ozone was found to induce reductions in CO2 assimilation and water use efficiency in the leaves of this species. These reductions were mainly related to a decline in the in vivo CO2 fixation capacity of Rubisco (V(c,max)), rather than to stomatal limitations or photoinhibitory damage (F(v):F(m)). In addition to chloroplast degeneration, other observed effects were callose accumulation, formation of pectinaceous wart-like cell wall exudates and phloem alterations. Moreover, ozone exposure significantly reduced root dry biomass. The possible relevance of these adverse effects for Mediterranean forbs is commented. These results show that endemic plants can be very sensitive to ozone, suggesting that risks associated with this pollutant should be taken into account for conservation purposes.

Responses of evergreen and deciduous Quercus species to enhanced ozone levels.

Plants of one evergreen oak (Quercus ilex) and three deciduous oaks (Q. faginea, with small leaves; Q. pyrenaica and Q. robur, with large leaves) were exposed both to filtered air and to enhanced ozone levels in Open-Top Chambers. Q. faginea and Q. pyrenaica were studied for the first time. Based on visible injury, gas exchange, chlorophyll content and biomass responses, Q. pyrenaica was the most sensitive species, and Q. ilex was the most tolerant, followed by Q. faginea. Functional leaf traits of the species were related to differences in sensitivity, while accumulated ozone flux via stomata (POD1.6) partly contributed to the observed differences. For risk assessment of Mediterranean vegetation, the diversity of responses detected in this study should be taken into account, applying appropriate critical levels.

Two Trebouxia algae with different physiological performances are ever-present in lichen thalli of Ramalina farinacea. Coexistence versus competition?

Ramalina farinacea is an epiphytic fruticose lichen that is relatively abundant in areas with Mediterranean, subtropical or temperate climates. Little is known about photobiont diversity in different lichen populations. The present study examines the phycobiont composition of several geographically distant populations of R. farinacea from the Iberian Peninsula, Canary Islands and California as well as the physiological performance of isolated phycobionts. Based on anatomical observations and molecular analyses, the coexistence of two different taxa of Trebouxia (working names, TR1 and TR9) was determined within each thallus of R. farinacea in all of the analysed populations. Examination of the effects of temperature and light on growth and photosynthesis indicated a superior performance of TR9 under relatively high temperatures and irradiances while TR1 thrived at moderate temperature and irradiance. Ramalina farinacea thalli apparently represent a specific and selective form of symbiotic association involving the same two Trebouxia phycobionts. Strict preservation of this pattern of algal coexistence is likely favoured by the different and probably complementary ecophysiological responses of each phycobiont, thus facilitating the proliferation of this lichen in a wide range of habitats and geographic areas.

Fungal-associated NO is involved in the regulation of oxidative stress during rehydration in lichen symbiosis.

BACKGROUND: Reactive oxygen species (ROS) are normally produced in respiratory and photosynthetic electron chains and their production is enhanced during desiccation/rehydration. Nitric oxide (NO) is a ubiquitous and multifaceted molecule involved in cell signaling and abiotic stress. Lichens are poikilohydrous organisms that can survive continuous cycles of desiccation and rehydration. Although the production of ROS and NO was recently demonstrated during lichen rehydration, the functions of these compounds are unknown. The aim of this study was to analyze the role of NO during rehydration of the lichen Ramalina farinacea (L.) Ach., its isolated photobiont partner Trebouxia sp. and Asterochloris erici (Ahmadjian) Skaloud et Peksa (SAG 32.85 = UTEX 911). RESULTS: Rehydration of R. farinacea caused the release of ROS and NO evidenced by the fluorescent probes DCFH2-DA and DAN respectively. However, a minimum in lipid peroxidation (MDA) was observed 2 h post-rehydration. The inhibition of NO in lichen thalli with c-PTIO resulted in increases in both ROS production and lipid peroxidation, which now peaked at 3 h, together with decreases in chlorophyll autofluorescence and algal photobleaching upon confocal laser incidence. Trebouxia sp. photobionts generate peaks of NO-endproducts in suspension and show high rates of photobleaching and ROS production under NO inhibition which also caused a significant decrease in photosynthetic activity of A. erici axenic cultures, probably due to the higher levels of photo-oxidative stress. CONCLUSIONS: Mycobiont derived NO has an important role in the regulation of oxidative stress and in the photo-oxidative protection of photobionts in lichen thalli. The results point to the importance of NO in the early stages of lichen rehydration.

Ozone-induced reductions in below-ground biomass: an anatomical approach in potato.

Potato plants were grown in open-top chambers under three ozone concentrations during two complete cropping seasons (93 and 77 d in 2004 and 2005, respectively). The effects of chronic exposure to ozone on leaf anatomy, cell ultrastructure and crop yield were studied. Severe cell damage was found, even at ambient ozone levels, mainly affecting the spongy parenchyma and areas near the stomata. Damage to the cell wall caused loss of cell contact, and loss of turgor pressure due to tonoplast disintegration, contributed to cell collapse. Phloem sieve plates were obstructed by callose accumulation, and damaged mesophyll cells increased their starch stores. Tuber yield fell sharply (24-44%), due to the biggest tubers becoming smaller, which affected commercial yield. These anatomical findings show the mechanisms of ozone effect on assimilate partitioning, and thus crop yield decrease, in potato. Further implications of ozone causing reductions in below-ground biomass are also discussed.