Francisco J. García-Breijo

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.

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.

Morphology, anatomy and germination response of heteromorphic achenes of Anthemis chrysantha J. Gay (Asteraceae), a critically endangered species.

This study demonstrates that Anthemis chrysantha , a ‘Critically Endangered’ annual plant, produces two morphs of achenes: white and dark achenes, which differ in size, mass, anatomy and germination behaviour. Fresh white achenes germinated at all temperatures assayed from 10 to 25°C in both continuous darkness and 12-h photoperiod, ranging between 24% at 25°C in darkness and 89% at 12/20°C in light, whereas fresh dark achenes did not germinate under any temperature or light conditions. To identify differences in dormancy type between the two morphs, germination of dry-stored achenes, and achenes stratified at 5 or 25°C for 2 months were tested in both darkness and light at 5, 15 and 12/20°C for dry-stored and warm-stratified (25°C) achenes; and at 15, 25 and 12/20°C for cold-stratified (5°C) achenes. Of the white achenes, 90% germinated during the cold stratification period. In general, dry storage and warm stratification did not increase germination compared to fresh achenes. However, dark achenes did not germinate under any conditions. Dark achene dormancy was only broken by mechanical scarification or by excising the embryo (germination reached 71%). An anatomical study showed that the mesocarp of dark achenes had no intercellular spaces and was much thicker and stronger than that of white achenes, making the entry of water difficult, and also preventing germination by mechanical restriction. This study demonstrated that dormancy in the dark achenes is likely caused by the thickness of their pericarp, physically impeding germination and hampering imbibition of water.

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.

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. Microsc. Res. Tech. 2011.

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.