Francisco Gasulla

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

BackgroundReactive 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).ResultsRehydration 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.ConclusionsMycobiont 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.

Oxidative stress induces distinct physiological responses in the two Trebouxia phycobionts of the lichen Ramalina farinacea

BACKGROUND AND AIMS Most lichens form associations with Trebouxia phycobionts and some of them simultaneously include genetically different algal lineages. In other symbiotic systems involving algae (e.g. reef corals), the relative abundances of different endosymbiotic algal clades may change over time. This process seems to provide a mechanism allowing the organism to respond to environmental stress. A similar mechanism may operate in lichens with more than one algal lineage, likewise protecting them against environmental stresses. Here, the physiological responses to oxidative stress of two distinct Trebouxia phycobionts (provisionally named TR1 and TR9) that coexist within the lichen Ramalina farinacea were analysed. METHODS Isolated phycobionts were exposed to oxidative stress through the reactive oxygen species propagator cumene hydroperoxide (CuHP). Photosynthetic pigments and proteins, photosynthesis (through modulated chlorophyll fluorescence), the antioxidant enzymes superoxide dismutase (SOD) and glutathione reductase (GR), and the stress-related protein HSP70 were analysed. KEY RESULTS Photosynthetic performance was severely impaired by CuHP in phycobionts, as indicated by decreases in the maximal PSII photochemical efficiency (F(v)/F(m)), the quantum efficiency of PSII (Φ(PSII)) and the non-photochemical dissipation of energy (NPQ). However, the CuHP-dependent decay in photosynthesis was significantly more severe in TR1, which also showed a lower NPQ and a reduced ability to preserve chlorophyll a, carotenoids and D1 protein. Additionally, differences were observed in the capacities of the two phycobionts to modulate antioxidant activities and HPS70 levels when exposed to oxidative stress. In TR1, CuHP significantly diminished HSP70 and GR but did not change SOD activities. In contrast, in TR9 the levels of both antioxidant enzymes and those of HSP70 increased in response to CuHP. CONCLUSIONS The better physiological performance of TR9 under oxidative conditions may reflect its greater capacity to undertake key metabolic adjustments, including increased non-photochemical quenching, higher antioxidant protection and the induction of repair mechanisms.

Suitability of chloroplast LSU rDNA and its diverse group I introns for species recognition and phylogenetic analyses of lichen-forming Trebouxia algae

To date, species identification of lichen photobionts has been performed principally on the basis of microscopic examinations and molecular data from nuclear-encoded genes. In plants, the chloroplast genome has been more readily exploited than the nuclear genome for systematic investigations. At the present time, very little information is available about the chloroplast genome of lichen-forming algae. For this reason, we have sequenced a portion of the gene encoding for the chloroplast large sub-unit rRNA (LSU rDNA) as a new molecular marker. Sequencing of the chloroplast LSU rDNAs revealed the existence of an unusual diversity of group I introns (a total of 31) within 15 analyzed Trebouxia species. The number, sequence and insertion site of these introns were very different among species, contributing to their recognition. A relatively large intron-free portion of the chloroplast LSU rDNA and part of the nuclear ribosomal cistron (18S-5.8S-26S) between the nuclear internal transcribed spacers (nrITS) were subjected to phylogenetic analyses. The obtained results indicate that data combination from both nuclear and chloroplast sequences can improve phylogenetic accuracy. Herein, we propose the suitability of both intronic and exonic sequences of the chloroplast LSU rDNA for species recognition, and an exonic sequence spanning from position 879 to 1837 in the Escherichia coli 23S rDNA for phylogenetic analyses of Trebouxia phycobionts.

The organic air pollutant cumene hydroperoxide interferes with NO antioxidant role in rehydrating lichen

Organic pollutants effects on lichens have not been addressed. Rehydration is critical for lichens, a burst of free radicals involving NO occurs. Repeated dehydrations with organic pollutants could increase oxidative damage. Our aim is to learn the effects of cumene hydroperoxide (CP) during lichen rehydration using Ramalina farinacea (L.) Ach., its photobiont Trebouxia spp. and Asterochloris erici. Confocal imaging shows intracellular ROS and NO production within myco and phycobionts, being the chloroplast the main source of free radicals. CP increases ROS, NO and lipid peroxidation and reduces chlorophyll autofluorescence, although photosynthesis remains unaffected. Concomitant NO inhibition provokes a generalized increase of ROS and a decrease in photosynthesis. Our results suggest that CP induces a compensatory hormetic response in Ramalina farinacea that could reduce the lichens antioxidant resources after repeated desiccation-rehydration cycles. NO is important in the protection from CP.

Photosynthesis in Lichen: Light Reactions and Protective Mechanisms

Lichens are symbiotic associations (holobionts) established between fungi (mycobionts) and certain groups of cyanobacteria or unicellular green algae (photobionts). This symbiotic association has been essential in establishing the colonization of terrestrial and consequently dry habitats. About 44 genera of algae and cyanobacteria have been reported as lichen photobionts. Due to the uncertain taxonomy of many of these photobionts, these numbers were considered as approximations only. Ahmadjian (1993) estimates that only 25 genera were typical lichen photobionts. The most common cyanobionts are Nostoc, Scytonema, Stigonema, Gloeocapsa, and Calothrix, in order of frequency (Budel, 1992). Green algal photobionts include Asterochloris, Trebouxia, Trentepohlia, Coccomyxa, and Dictyochloropsis (Gartner, 1992). These authors assessed that more than 50% of all lichen species are associated with Trebouxia and Asterochloris species. However, this is just estimation since in only 2% of the described lichen species the photobiont genus is reported (Tschermak-Woess, 1989), mostly by the difficulties to isolate and then characterize the algae from the lichen thalli. Lichens are well known for their slow growth and longevity. Their radial growth is measured in millimetres per year (Hale, 1973), while individual lichens live for hundreds or even thousands of years. It is assumed that in lichens the photobiont population is under mycobiont control. Lichenologists have proposed some control mechanisms such as, cell division inhibitors (Honegger, 1987), phytohormones (Backor & Hudak, 1999) or nutrients competition (Crittenden et al., 1994; Schofield et al., 2003). Similar to plants, all lichens photosynthesise. They need light to provide energy to make their own matter. More specifically, the algae in the lichen produce carbohydrates and the fungi take those carbohydrates to grow and reproduce. The amount of light intensity needed for optimal lichen growth varies widely among species. The optimum light intensity range of most algal photobionts in axenic cultures is very low, between 16-27 μmol m-2 s-1. If the response of cultured photobionts to light is similar to that of the natural forms (lichen), then there must be additional mechanisms protecting the algae in the lichen that are not developed under culture conditions. Pigments and crystal of secondary metabolites in the

Chlororespiration induces non-photochemical quenching of chlorophyll fluorescence during darkness in lichen chlorobionts

Lichens and their algal partners are desiccation-tolerant organisms and as such survive after the complete loss of water. This trait is the consequence of several physiological, biochemical and structural features, including specific mechanisms dissipating excess light to avoid photooxidative stress. The maximum quantum yield of photosystem II (PSII; Fv /Fm ) is widely used as a sensitive indicator of photosynthetic performance and is calculated after complete relaxation in darkness of the fluorescence quenching associated with active light energy dissipation mechanisms. Unexpectedly, we observed that lichens and isolated chlorobionts (chlorophyte symbionts in lichen) maintained in darkness for several hours showed a strong decrease in the ratio Fv /Fm , which was reversible after re-illumination. We analyzed this dark-induced Fv /Fm decay in the chlorobiont Asterochloris erici through steady-state and fast-induction kinetics of chlorophyll a fluorescence and simultaneous P700 oxidation measurements. We found that the gradual decay of Fv /Fm in darkness was caused by reversible dark-induced inactivation of some PSII reaction centers that was accompanied by a decrease in the flux of electrons to PSI. Darkness induced the plastoquinone-reductase activity associated with chlororespiration and the phosphorylation of light harvesting complex (LHC). We propose that upon phosphorylation the LHC detaches from PSII, resulting in a decrease of exciton-trapping by PSII reaction centers and, consequently, an increased dissipation of light energy. This mechanism probably serves an ecophysiological function in lichens to prevent the damage at dawn or under strong fluctuating light conditions when lichens are in a hydrated state.