Abscisic acid and polyamine contents in the lichens Pseudevernia furfuracea and Ramalina farinacea

The levels of endogenous abscisic acid and total polyamine contents (putrescine and spermidine) were studied in two lichen species differing in desiccation tolerance. ABA and polyamine contents showed significant differences between air-dried and water-treated thalli of Pseudevernia furfuracea and Ramalina farinacea. The levels of putrescine (put) and spermidine (spd) in water-treated P. furfuracea thalli were found higher than in dry samples. The values for spd were almost threefold and for put almost 1.4-fold higher than in airdried samples. The levels of put and spd in water-treated R. farinacea were lower than in the air-dried samples. The lowest values for spd and put were almost 33-fold and 2.8-fold less, respectively. ABA contents of water-treated P. furfuracea and R. farinacea increased by 2.65% and 25.5% when compared with air-dried thallus. This text was submitted by the authors in English.     

A new method to isolate lichen algae by using Percoll®gradient centrifugation

Abstract:A rapid method to isolate intact functional algae from the lichens Evernia prunastri and Ramalina farinacea has been developed. This method is based on the use of Percoll^®gradients after mechanical disruption of lichen thalli. Results obtained show that the algal preparations were virtually free of contamination by fungal hyphae. The purified algal cells were photosynthetically active and without symptoms of photoinhibition, which indicates their functional integrity. This method may be used for the isolation of intact algae from a broad range of lichen species.     

The Adaptation of Plankton Algae

The various aspects of the adaptation of plankton algae lo light and temperature are discussed. The shape of a light intensity-photosynthesis curve is shown to be an important means of describing the physiological adjustment of an algal population. If the algae are not exposed to adverse influences such as poisons, pronounced nutrient deficiency or light shocks, the rate of real photosynthesis per mg chlorophyll a at 1 Klux (incandescent light) should be about 0.4–0.6 mg C/hour. Hence this rate presents an excellent means of judging the quality of experiments. Experiments are presented where Chlorella pyrenoidosa was adapted to light intensities between 0.32 klux and 21 Klux. This alga adapts to different light intensities by varying the amount of pigments per cell. Algae grown at 1 Klux have about 10 times more chlorophyll per cell than those grown at 21 klux. Other species of algae—but by no means all—are shown to behave in the same way. The problem of algal resistance to photo-oxidation at high light intensities is discussed. Adaplation is shown to he one of the mechanisms which make the algae resistent. “Chlorophyll inactivation” is another. Experiments with the diatom Skeletonema costatum concerning adaptation to different temperatures have been performed. The fact that the alga has essentially the same rate of photosynthesis per cell at all light intensities at 20°C and 7°C, may be attributed to an increase of all the enzymes at the low temperature. The amount of protein per cell was twice as high at 7°C as at 20°C.     

Symbiosis constraints: Strong mycobiont control limits nutrient response in lichens

Symbioses such as lichens are potentially threatened by drastic environmental changes. We used the lichen Peltigera aphthosa—a symbiosis between a fungus (mycobiont), a green alga (Coccomyxa sp.), and N₂‐fixing cyanobacteria (Nostoc sp.)—as a model organism to assess the effects of environmental perturbations in nitrogen (N) or phosphorus (P). Growth, carbon (C) and N stable isotopes, CNP concentrations, and specific markers were analyzed in whole thalli and the partners after 4 months of daily nutrient additions in the field. Thallus N was 40% higher in N‐fertilized thalli, amino acid concentrations were twice as high, while fungal chitin but not ergosterol was lower. Nitrogen also resulted in a thicker algal layer and density, and a higher δ¹³C abundance in all three partners. Photosynthesis was not affected by either N or P. Thallus growth increased with light dose independent of fertilization regime. We conclude that faster algal growth compared to fungal lead to increased competition for light and CO₂ among the Coccomyxa cells, and for C between alga and fungus, resulting in neither photosynthesis nor thallus growth responded to N fertilization. This suggests that the symbiotic lifestyle of lichens may prevent them from utilizing nutrient abundance to increase C assimilation and growth.     

Water Status of Green and Blue-green Phycobionts in Lichen Thalli after Hydration by Water Vapor Uptake: Do They Become Turgid?

It is known from previous investigations that dry lichens with green algae are able to recover net photosynthesis through rehydration with water vapor, whereas all blue-green lichens tested so far lack this ability. The REM micrographs of the present study show that the green phycobionts (Trebouxia spec.) of Ramalina maciformis become turgid only after water vapor uptake. In contrast, the blue-green phycobionts (Nostoc spec.) of Peltigera rufescens do not differ in appearance from the dry state, even when the thallus has reached equilibrium with the water vapor-saturated air; they require liquid water for turgidity. It is hypothesized that, after humidity hydration, water content is not sufficient for reestablishment of a functioning osmotic cell system in the blue-green phycobiont.     

Characterization of Enzymatic Antioxidants in the Lichen Ramalina lacera and Their Response to Rehydration

Lichens are slow-growing associations of fungi and green algae or cyanobacteria. This symbiotic association forms a common thallus that does not possess roots or a waxy cuticle and depends mainly on atmospheric input of mineral nutrients. The lifestyle of most lichens is composed of alternating periods of desiccation with low metabolic activity and hydration that induces increase in their metabolism. We have previously shown that rehydration of the naturally desiccated lichen Ramalina lacera resulted in a rapid increase in photosynthesis and was accompanied by a burst of intracellular production of reactive oxygen species and nitric oxide, as well as a transient decrease in water-soluble antioxidant capacity. We report here on enzymatic antioxidants of R. lacera and their response to rehydration. Native gel electrophoresis of crude extracts of R. lacera stained for superoxide dismutase (SOD) activity revealed four Fe-SOD and four Mn-SOD electromorphs that are synthesized by the alga, a Cu/Zn-SOD and a Mn-SOD that are the product of the fungus, and two catalases synthesized one by the fungus and the other by the algae. In addition, we detected glutathione reductase and glucose-6-phosphate dehydrogenase activities in crude extracts of R. lacera. Rehydration of the thalli resulted in a decrease in SOD activity of all forms, and a transient decrease in total catalase activity, as well as a decrease in the antioxidant auxiliary enzymes glutathione reductase and glucose-6-phosphate dehydrogenase.     

Effects of chromium VI stress on photosynthesis,chlorophyll integrity,cell viability,and proline accumulation in lichen <Emphasis Type="Italic">Ramalina farinacea</Emphasis>

In order to contribute to understanding of the response to metal stress, Ramalina farinacea (L.) Ach. was treated with different concentrations of chromium (VI) (5, 15, and 30 mM of K₂CrO₄) for 1, 3, and 24 h, and alterations in the photosystem II photosynthetic quantum yield, pigment content, integrity of chlorophyll, cell viability, and proline accumulation were investigated. Significant alterations of the photosynthetic quantum yield (F _v/F _m) ratio were observed in response to the increase in chromium concentration. The F _v/F _m ratio decreased in R. farinacea following 24-h treatment with 30 mM Cr⁶⁺ solution. In present study, in both control and other plant groups treated with 5 mM Cr⁶⁺, the Chl a/b ratio was approximately within the range of 2.0–3.5. However, this ratio significantly decreased for the samples treated with 15 (exposure period of 24 h) and 30 mM; (exposure periods of 3 and 24 h) Cr⁶⁺. We also showed that cell viability of samples treated with 15 and 30 mM Cr⁶⁺ significantly decreased. Accumulation of metal resulted in proline accumulation in R. farinacea thalli; however, when photodestructive effects on photosystem II occurred, proline intracellular concentration declined. On the basis of these results, we suggest that proline accumulation might not be the stress marker during heavy metal stress.     

Experimental Studies on the Physiology of the Phycobiont and Mycobiont Ramalina ecklonii By

The lichenized fungus and alga of the fruticose lichen Ramalini ecklonii were isolated into pure cultures. The ascospores of the fungus failed to germinate in less than five weeks incubation in spite of the use of a variety of cultural conditions. The fungus showed a considerable increase in growth on malt extract agar. Both organisms showed a marked tolerance for high concentrations of glucose although growth was quantitatively reduced. The fungus was able to use a variety of carbon and nitrogen sources as well as an extract of algal cells. Cultivation in the absence of biotin and thiamine failed to yield significant amounts of growth. The alga yielded 27 mg of dry weight after three weeks in a synthetic medium under low light intensities. The alga could be grown in satisfactory amounts on CO₂ and inorganic salts with moderate light intensities. Experiments using ¹⁴CO₂ showed the fungus able to incorporate the extra-cellular and intra-cellular products of algal metabolism. The rate of incorporation of extra-cellular products was inhibited by high concentrations of biotin and thiamine. The alga assimilated ^l4CO₂ which was retained by the cells over a period of 14 days, at which time 78 per cent of the activity was insoluble in 80 per cent ethanol. An extract of the fungus labelled with ¹⁴C glucose was partially taken up by the alga and 50 per cent of the label was insoluble in 80 per cent after three days incubation in the light. No lichen acids were found in either the fungal cultures or the algal cultures although large amounts (e.g. 2 liters) of material were extracted and chromatographed. Usnic acid was produced by the intact lichen thallus.     

Effect of low water potential on photosynthesis in intact lichens and their liberated algal components

Earlier experiments (T.D. Brock 1975, Planta124, 13–23) addressed the question whether the fungus of the lichen thallus might enable the algal component to function when moisture stress is such that the algal component would be unable to function under free-living conditions. It was concluded that the liberated phycobiont in ground lichen thalli could not photosynthesize at water potentials as low as those at which the same alga could when it was present within the thallus. However, our experience with lichen photosynthesis has not substantiated this finding. Using instrumentation developed since the mid-1970's to measure photosynthesis and control humidity, we repeated Brock's experiments. When applying “matric” water stress (equilibrium with air of constant relative humidity) we were unable to confirm the earlier results for three lichen species including one of the species,Letharia vulpina, had also been used by Brock. We found no difference between the effects of low water potential on intact lichens and their liberated algal components (ground thallus material and isolated algae) and no indication that the fungal component of the lichen symbiosis protects the phycobiont from the adverse effects of desiccation once equilibrium conditions are reached. The photosynthetic apparatus of the phycobiont alone proved to be highly adapted to water stress as it possesses not only the capability of functioning under extremely low degrees of hydration but also of becoming reactivated solely by water vapor uptake.     

Characterization of enzymatic antioxidants in the lichen Ramalina lacera and their response to rehydration

Lichens are slow-growing associations of fungi and green algae or cyanobacteria. This symbiotic association forms a common thallus that does not possess roots or a waxy cuticle and depends mainly on atmospheric input of mineral nutrients. The lifestyle of most lichens is composed of alternating periods of desiccation with low metabolic activity and hydration that induces increase in their metabolism. We have previously shown that rehydration of the naturally desiccated lichen Ramalina lacera resulted in a rapid increase in photosynthesis and was accompanied by a burst of intracellular production of reactive oxygen species and nitric oxide, as well as a transient decrease in water-soluble antioxidant capacity. We report here on enzymatic antioxidants of R. lacera and their response to rehydration. Native gel electrophoresis of crude extracts of R. lacera stained for superoxide dismutase (SOD) activity revealed four Fe-SOD and four Mn-SOD electromorphs that are synthesized by the alga, a Cu/Zn-SOD and a Mn-SOD that are the product of the fungus, and two catalases synthesized one by the fungus and the other by the algae. In addition, we detected glutathione reductase and glucose-6-phosphate dehydrogenase activities in crude extracts of R. lacera. Rehydration of the thalli resulted in a decrease in SOD activity of all forms, and a transient decrease in total catalase activity, as well as a decrease in the antioxidant auxiliary enzymes glutathione reductase and glucose-6-phosphate dehydrogenase.     

Photosynthetic acclimation to different light levels in the brown marine macroalga, <Emphasis Type="Italic">Hizikia fusiformis</Emphasis> (Sargassaceae,Phaeophyta)

Hizikia fusiformis thalli experience dynamic incident light conditions during the period of growth. The present study was designed to examine how changing photon irradiance affects the photosynthesis both in the short and long terms by culturing H. fusiformis under three different light levels: 35 μmol photons m^-2 s^-1 (low light, LL), 85 μmol photons m^-2 s^-1 (intermediate light, IL), and 165 μmol photons m^-2 s^-1 (high light, HL). A similar relative growth rate was observed between IL- and HL-grown algae, but the growth rate was significantly reduced in LL-grown algae. The photosynthetic rates (P _n) measured at their respective growth light levels were found to be lowest in the thalli grown at LL and highest at HL. However, LL-grown algae exhibited much higher P _n in comparison with IL- and the HL-grown thalli at the same measuring photosynthetic photon flux density, indicating the photosynthetic acclimation to low growth light in H. fusiformis. The photosynthesis–light curves showed that LL-grown algae had a highest light-saturating maximum P _n (P _max) in comparison with IL- or HL-grown algae when the photosynthetic rates were expressed on the biomass basis. However, P _max was highest in HL-grown algae compared to IL- or LL-grown algae when the rates were normalized to chlorophyll a. The photosynthesis–inorganic carbon (Ci) response curves were also significantly affected by the growth light conditions. The highest value of apparent photosynthetic conductance occurred in LL-grown algae while the lowest value in HL-grown algae. Additionally, the activity of external carbonic anhydrase (CA) tended to increase while the total CA activity inclined to decrease in H. fusiformis thalli when the growth light level altered from 35 to 165 μmol photons per square meter per second. The external CA inhibitors showed a higher inhibition in HL-grown algae compared with LL-grown algae. It was proposed that photosynthetic acclimation to low light condition in H. fusiformis was achieved through an increase in the number of reaction centers and increased capacities of electron transport and of Ci transport within cells. The ability of photosynthetic acclimation to low light confers H. fusiformis thalli to overcome the environmental low light condition as a result of the attenuation of seawater or self-shading through enhancing its photosynthetic performance and carbon assimilation necessary for growth.     

Relationship between carbohydrate movement and the symbiosis in lichens with green algae

Summary When isolated in pure culture, four genera of lichen algae were able to produce the polyol which is known to move from the alga to the fungus in lichens with these algae. This conclusion corrects earlier suggestions that the mobile polyol is only formed by the alga in the lichen thallus. Stichococcus produced sorbitol and it is therefore suggested that, in lichens with this alga, sorbitol moves between the symbionts. Hyalococcus and Stichococcus had a similar pattern of incorporation of H¹⁴CO ₃ ^- in the light, suggesting a close relationship between these algae which are only separated now on morphological grounds.The pattern of incorporation of H¹⁴CO ₃ ^- in the light into Cladonia cristatella and its alga (Trebouxia erici) in culture indicates that in the cultured algae more ¹⁴C was incorporated into ethanol insoluble substances and lipids and less into ribitol than in the lichen. The pattern in a joint culture of the alga and the fungus of C. cristatella was approximately intermediate between that of the lichen and the alga. However, only a small amount of ¹⁴C fixed by the alga reached the fungus in the joint culture, and it is therefore suggested that the presence of the fungus without morphological differentiation into a lichen thallus is not sufficient to promote the alga to release carbohydrate.     

Simple method for fractionation of <Emphasis Type="Italic">Parmelia sulcata</Emphasis> lichen thalli

Three fractions were isolated from Parmelia sulcata lichen thalli: mycobiont-enriched fraction, highly purified preparation of Trebouxia sp. alga, and a photobiont-enriched fraction, in which the fungal component and symbiotic relations between the bionts were retained. The ratio between the fungal and algal components in all fractions was estimated from the photosynthesis/respiration and chlorophyll/dry weight ratios. Simple methods for lichen thallus fractionation are suggested to study symbiotic relationships of bionts.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 306–310.Original Russian Text Copyright © 2005 by Maslov, Pavlova.This revised version was published online in April 2005 with a corrected cover date.     

Photosynthetic CO2-use efficiency in lichens and their isolated photobionts: the possible role of a CO2-concentrating mechanism

The CO₂ dependence of net CO₂ assimilation was examined in a number of green algal and cyanobacterial lichens with the aim of screening for the algal/cyanobacterial CO₂-concentrating mechanism (CCM) in these symbiotic organisms. For the lichens Peltigera aphthosa (L.) Willd., P. canina (L.) Willd. and P. neopolydactyla (Gyeln.) Gyeln., the photosynthetic performance was also compared between intact thalli and their respective photobionts, the green alga Coccomyxa PA, isolated from Peltigera aphthosa and the cyanobacterium Nostoc PC, isolated from Peltigera canina. More direct evidence for the operation of a CCM was obtained by monitoring the effects of the carbonic-anhydrase inhibitors acetazolamide and ethoxyzolamide on the photosynthetic CO₂use efficiency of the photobionts. The results strongly indicate the operation of a CCM in all cyanobacterial lichens investigated and in cultured cells of Nostoc PC, similar to that described for free-living species of cyanobacteria. The green algal lichens were divided into two groups, one with a low and the other with a higher CO₂-use efficiency, indicative of the absence of a CCM in the former. The absence of a CCM in the low-affinity lichens was related to the photobiont, because free-living cells of Coccomyxa PA also apparently lacked a CCM. As a result of the postulated CCM, cyanobacterial Peltigera lichens have higher rates of net photosynthesis at normal CO₂ compared with Peltigera aphthosa. It is proposed that this increased photosynthetic capacity may result in a higher production potential, provided that photosynthesis is limited by CO₂ under natural conditions.     

The Adaptation of Plankton Algae

The variation in Skeletonema cells grown at 3 klux continuous illumination and 20°C is reported. Four different types of lamps gave no difference in the photosynthetic characteristics. The average diameter of the cells decreased from 8–3.5 μ during their six months vegetative period. The ratio between the pigment content in the largest and the smallest cells was about 2:1. A good correlation between cell volume and chlorophyll a content was found for this species. The content of chlorophyll c generally varied between 4 and 17 per cent of the chlorophyll a content. — A distinct correlation between the chlorophyll a content and the rate of photosynthesis per unit of cells at low light intensity was found. The rate of photosynthesis, in mg C per mg chlorophyll a and hour at 1 klux, varied between 0.40 and 0.70 for all 60 experiments with an average value of 0.56. The corresponding value for cells deficient in phosophorus was 0.19 and for cells deficient in nitrogen 0.09. — The material also showed a good correlation between the rate of photosynthesis per cell at 1 klux and the light-saturated rate of photosynthesis. I_k varied between 7 and 13 klux.     

Obtaining plantlets from apical meristem of the red alga Gelidium sp.

For the first time, plantlets were obtained from fragments and cell aggregates (CA) of apical meristem of the red alga Gelidium sp. After two months of cultivation, an initial weight of 100 mg of fragments and CA from fresh meristem produced 3 g of plantlets without rhizoids. During the same period of cultivation, 100 mg of meristem fragments and CA isolated from thalli by the freezing-thawing procedure produced more than 20 g of plantlets with rhizoids. It is assumed that our methods for obtaining plantlets from fragments and CA of fresh and frozen-thawed meristem could be used to generate mass planting material for cultivation of algae (plantlets with rhizoids) in the sea and for tank-bubbling cultivation (plantlets without rhizoids). We speculate that meristem cells of frozen-thawed algae might be natural “seedstock” in the Arctic and Antarctic seas.     

Lichen protein affinity towards walls of cultured and freshly isolated phycobionts and its relationship to cell wall cytochemistry

Abstract The binding capacity of protein fractions ( M _r > 8000) from five lichens, Hypocenomyce scalaris, Hypogymnia physodes, Ramalina fastigiata, Umbilicaria pustulata and Xanthoria parietina was tested towards their freshly isolated phycobionts and towards ten cultures of phycobionts, five of which were from the above-mentioned lichens. The protein fractions from H. scalaris, R. fastigiata, U. pustulata and X. parietina bind intensely to a cultured Pseudotrebouxia from H. physodes and a cultured Trebouxia from X. parietina , but not to the other cultured algae, and not to the freshly isolated algae. A protein fraction between M _r 3500 and 8000 from H. physodes shows a faint binding to its own cultured algae. The nature of the binding and its relevance in connection with the mutual recognition of alga and fungus are briefly discussed.     

ASCOPHYLLUM NODOSUM (PHAEOPHYTA) IN AXENIC CULTURE AND ITS RESPONSE TO THE ENDOPHYTIC FUNGUS MYCOSPHAERELLA ASCOPHYLLI AND EPIPHYTIC BACTERIA1

Ascophyllum nodosum (L.) Le Jol. was freed from bacteria and the endophytic fungus Mycosphaerella ascophylli Cotton by repeated treatment with chlorine solutions and grown in artificial seawater. Two types of axenic culture of different origin were obtained. Type 1 was developed from apices of A. nodosum collected in the sea. Type 2 was from plants which developed from adventitious embryos on rhizoids formed by type 1. This is the first time A. nodosum has been cultivated axenically. Growth of the axenic alga was increased by IAA, 21P and zeatin. Without external growth regulators some strains of the axenic alga deteriorated within a year; others developed a filamentous habit. Sulfur in a reduced state also stimulated growth. Addition of either glucose, mannose or mannitol to the medium caused the formation of calluslike layers of loosely packed colorless cells under the epidermis of the thalli and the epidermis was sloughed off. No increase in thallus length was noticed. Mycosphaerella ascophylli in axenic culture did not excude any substances stimulating growth of the alga, but that does not exclude an influence of the fungus on the alga in vivo. The fungus, when growing within the alga, seemed to have little influence on algal morphology. A bacterized but fungus-free A. nodosum was cultivated in an artificial seawater for 8 years. In the bacteria-free alga, the fungus protruded from the epidermis and evidently utilized the alga as a carbon source. The bacteria thus seem much more important than the fungus for normal growth of the Ascophyllum plant.     

THE DIFFERENTIAL ACTION OF CHEMICAL AGENTS,ESPECIALLY POLYMYXIN-B,ON CERTAIN ALGAE,BACTERIA AND FUNGI

Galloway , R. A., and R. W. Krauss (U. Maryland, College Park.) The differential action of chemical agents, especially polymyxin-B, on certain algae, bacteria and fungi. Amer. Jour. Bot. 46(1): 40-49. Illus. 1959.—Representatives from each of the major groups of compounds exhibiting toxicity toward microorganisms were screened to determine their relative effect on 2 green algae, Scenedesmus obliquas and Chlorella pyrenoidosa, 1 blue green alga, Anabaena variabilis, 4 species of bacteria in the genera Flavobacterium, Achromobacter, and Pseudomonas, and the fungus Aspergillus sydowii. The objective was the determination of the differential toxicity shown by chemical agents against these microorganisms. Polymyxin-B sulfate proved to have a high degree of selectivity which warranted detailed examination of its action. Manometric studies were made of the effects of this compound on rates of photosynthesis and respiration. The rate of photosynthesis in Scenedesmus was only slightly diminished by 640 p.p.m. polymyxin-B, but a rapid reduction in rate to a very low level was observed in Chlorella. The endogenous respiration rate in Scenedesmus was more than doubled. The rate of respiration on glucose, pyruvate, fumarate, and acetate remained unchanged except for a slight transient rise at a low pH or in the absence of magnesium. The effect of 640 p.p.m. of polymyxin-B on photosynthesis of Chlorella and on respiration, whether endogenous or on any of the substrates mentioned above, was a rapid, permanent reduction to a low level. Similar reduction in rate of respiration at 160 p.p.m. was observed in the bacteria and fungus, when these organisms were utilizing lactose, pyruvate, or fumarate as substrates. Two of the bacteria were exceptions in that no reduction occurred when they were respiring lactose. Inhibition of glucose phosphorylation was not found in the algae. Therefore some system other than an alternative phosphorylation must account for resistance to polymyxin-B.