Photochromic pigments in akinetes and pigment characteristics of akinetes in comparison with vegetative cells of Anabaena variabilis

Since akinete germination is triggered by light and the action spectrum for this process has features in common with the spectra of the two photochromic pigments, phycochromes b and d, a search was made for the presence of these phycochromes in akinetes of the blue-green alga. Anabaena variabilis Kützing. Allophycocyanin-B was also looked for, since the action spectrum for akinete germination points to a possible participation of this pigment too. Isoelectric focusing was used for purification of the pigments. The different fractions were investigated for phycochromes b and d by measuring the absorbance difference spectra: for phycochrome b. 500 nm irradiated minus 570 nm irradiated, and for phycochrome d, 650 nm irradiated minus 610 nm irradiated. For determination of allophycocyanin-B. fourth derivative analysis of absorption spectra was made for some of the fractions from the isoelectric focusing column. Phycochrome b was also assayed for by measuring in vivo absorption difference spectra. The assays were positive for all three pigments. The complete photosynthetic pigment systems were also studied by in vivo fluorescence measurements on both akinetes and vegetative cells of Anabaena variabilis. Fluorescence emission and excitation spectra at selected emission wavelengths were measured at room temperature and liquid nitrogen temperature. The energy transfer from phycoerythrocyanin to phycocyanin is very efficient under all conditions, as is the energy transfer from phycocyanin to allophycocyanin at room temperature. At low temperature, however, phycocyanin is partly decoupled from allophycocyanin, particularly in the akinetes; the energy transfer from allophycocyanin to chlorophyll a is less efficient at low temperature in both types of cells, but especially in akinetes. Delayed light emission was measured for both types of cells and found to be very weak in akinetes compared to vegetative cells. From this study it would seem that akinetes lack an active photosystem II, although the 691 nm peak in the 570 nm excited low temperature fluorescence emission spectrum proves the presence of photosystem II chlorophyll, and also its energetic connection to the phycobilisomes.     

Effects of UV-light andγ-rays on the survival,akinete formation and akinete germination inStigeoclonium pascheri

The damage produced by UV light to any of the three different stages of the life cycle of the parent generation of the green algaS. pascheri, i.e. akinetes, germinating akinetes and vegetative cells remained up to the stage of germination of akinetes of the first generation and no deleterious effect was reported thereafter. Lower doses of γ-rays (25–75 Gy) increased the percentage germination of akinetes and germinating akinetes of parent generation. The percentage germination of akinetes, germinating akinetes, survival of colonies originated from vegetative cells and sporulation of cells of the parent generation decreased with increasing doses from 100 to 300 Gy. The γ-induced effect to any of the three different stages was not transferred to the subsequent stage of algal generation.     

UV‐A/BLUE LIGHT–INDUCED REACTIVATION OF SPORE GERMINATION IN UV‐B IRRADIATED ULVA PERTUSA (CHLOROPHYTA)1

Recent reduction in the ozone shield due to manufactured chlorofluorocarbons raised considerable interest in the ecological and physiological consequences of UV‐B radiation (λ=280–315 nm) in macroalgae. However, early life stages of macroalgae have received little attention in regard to their UV‐B sensitivity and UV‐B defensive mechanisms. Germination of UV‐B irradiated spores of the intertidal green alga Ulva pertusa Kjellman was significantly lower than in unexposed controls, and the degree of reduction correlated with the UV doses. After exposure to moderate levels of UV‐B irradiation, subsequent exposure to visible light caused differential germination in an irradiance‐ and wavelength‐dependent manner. Significantly higher germination was found at higher photon irradiances and in blue light compared with white and red light. The action spectrum for photoreactivation of germination in UV‐B irradiated U. pertusa spores shows a major peak at 435 nm with a smaller but significant peak at 385 nm. When exposed to December sunlight, the germination percentage of U. pertusa spores exposed to 1 h of solar radiation reached 100% regardless of the irradiation treatment conditions. After a 2‐h exposure to sunlight, however, there was complete inhibition of germination in PAR+UV‐A+UV‐B in contrast to 100% germination in PAR or PAR+UV‐A. In addition to mat‐forming characteristics that would act as a selective UV‐B filter for settled spores under the parental canopy, light‐driven repair of germination after UV‐B exposure could explain successful continuation of U. pertusa spore germination in intertidal settings possibly affected by intense solar UV‐B radiation.     

Environmental influences on akinete germination and development in Nodularia spumigena (Cyanobacteriaceae), isolated from the Gippsland Lakes, Victoria, Australia

This study was carried out to investigate the genesis of N. spumigena blooms by specifically studying the effects of environmental variables (salinity, nitrogen, phosphorus and light) on the germination of N. spumigena akinetes. Optimal conditions for maximum germination and germling growth were determined by exposing akinetes to a range of salinities and nutrient (nitrogen and phosphorus) concentrations under two different irradiances. At pre-determined time periods, treatments were sampled and the percent germination and length of germlings assessed. The results indicated that akinete germination and germling growth were optimal at salinities from 5 to 25 and significantly reduced outside this range. A positive correlation in germination was observed with increasing nutrient (phosphorus and nitrate) concentration. Similarly, germling growth increased with increasing concentrations of both nutrients. Irradiance significantly influenced both germination and growth during salinity experiments, whereas in nutrient addition experiments, irradiance had no effect on germination; however, growth was significantly influenced during phosphorus addition experiments. Consequently, salinity and light appeared to be most critical in the germination process for N. spumigena akinetes, with phosphorus most important for germling growth. The study showed that N. spumigena may be able to germinate under environmental conditions outside its optimal range, but the growth of the germling is significantly reduced, which in turn suggests that its ability to form a bloom outside its optimal environmental conditions would also be greatly reduced.     

Effects of salinity and source of inocula on germination of Anabaena akinetes from a tidally influenced lake

1. Sedimentary akinetes (resting stages) may represent significant potential inocula for nuisance blooms of cyanobacteria. We studied the effects of salinity and sediment source on the germination and subsequent growth of Anabaena flos‐aquae akinetes from a shallow, tidally influenced lake. 2. Surface sediments collected from littoral and open‐water sites were used as inocula to culture A. flos‐aquae akinetes in four salinities (0.1, 2.2, 4.4 and 6.5) over 22 days. Akinete germination and development was followed by counting developmental stages every second day. 3. Filament growth, but not akinete germination, was inhibited by salinity and there were significantly fewer filaments at 6.5 than at 0.1 and 2.2. Cultures inoculated with littoral sediment had more akinetes, germlings and filaments than those inoculated with open‐water sediment. 4. Sediment is a potential source of inocula for Anabaena blooms in the lake, which potentially could develop solely from this source because germination and subsequent filament growth do not depend on the existence of an initial pelagic Anabaena population.     

Influence of Environmental Stress on the Germination of Anabaena vaginicola Akinetes

Germination of akinetes of Anabeana vaginicola v. fertilissimaPrasad in response to environmental stress was studied. Additionof nitrate to the medium induced early and maximum germination(96 per cent), whereas less than half of the akinetes germinatedwhen either nitrate or phosphate was omitted from the medium.The pH range over which germination occurred was 7.0–9.0.The desiccated akinetes after rehydration germinated after acertain lag period, depending upon the dehydration state. Thetemperature optimum for germination and vegetative growth wasthe same (25 °C) and germination did not occur at 5 °Cor above 35 °C. The limit of heat shock tolerated was 55°C for 4 min. In addition to white light, only the red partof the visible spectrum induced germination. Ultraviolet radiationreduced germination rate presumably by inducing thymine dimersin DNA. The photoreactivating system (s) in akinetes is certainlynon-photosynthetic. LD₅₀ photon flux densities were 300 Jm^–2for akinetes and 240 Jm^–2 for vegetative cells. Anabaena vaginicola, blue-green alga, akinete, germination, environmental stress     

Influence of certain environmental factors on spore germination and spore differentiation inPithophora oedogonia

Of the light- and dark-stored spores ofPithophora oedogonia for over a period of one and half years, the latter exhibited complete failure of germination under controlled culture conditions. pH 8, blue light and 1 klx intensity of white light proved to be ideal conditions as they resulted in optimum germination of akinetes. pH 5, green light and 0.25 klx light intensity delayed the initiation of sporulation and also reduced percentage sporulation. All filaments died before sporulation in pH 4.     

Vegetative survival, akinete formation and germination in three blue-green algae and one green alga in relation to light intensity, temperature, heat shock and UV exposure

The mere vegetative survival was not sufficient but suitable growth conditions were required for akinete formation to occur in the blue-green algaeAnabœna iyengarii, Westiellopsis prolifica, Nostochopsis lobatus and in the green algaPithophora oedogonia. In all algae, akinetes were neither formed nor germinated in darkness, and while dim light of 300 lx was sufficient for most of akinetes to germinate and also to maintain vegetative survival, it was not adequate for optinum akinete formation. Although akinetes of all algae could germinate at 35°C, both the vegetative survival and akinete formation were markedly suppressed at this temperature. Heat or UV shock of any level, whether ineffective or effecting vegetative survival, did not promote akinete formation or germination in any alga tested. Akinetes of all algae under study were relatively tolerant to heat and also to some extent to UV. Both wet and dried akinetes of all algae were equally UV tolerant. In all algae, the viability of both wet and dried akinetes decreased more or less equally with storage time, but the decrease was more drastic when storage temperature was progressively lowered from 20 to 0°C. Hence the akinetes can tolerate dryness but not frost.     

Energetic and metabolic requirements for the germination of akinetes of the cyanobacteriumNostoc PCC 7524

Although akinetes ofNostoc PCC 7524 lost little of their main photosynthetic pigments, phycocyanin and chlorophyll, with increasing age after the onset of sporulation, they lost at least 90% of their photosynthetic and respiratory capacities. Germination needed the supply of light throughout the process, though previous dark metabolism accelerated the following light process. In standard conditions, both respiratory and photosynthetic capacities increased markedly during the first 9–10 h, a time sufficient for the first doublets to appear, but when pigment contents had not yet changed. However, while respiratory capacity could be reacquired without de nove metabolism, resumption of photosynthetic capacity needed RNA and protein synthesis. The energetic requirement for germination was not efficiently fulfilled by cyclic photosynthesis on PSI alone or respiration alone. In the presence of both PSI and respiratory activities only 21% of the akinetes germinated, their endogenous carbon reserves thus being inadequate to support the process to completion. The addition of sucrose to such cultures permitted all of the akinetes to germinate, but at a very slow rate. Rapid and complete germination was only observed when both photosystem operated.Abbreviations Chl Chlorophyll - Phy phycocyanin - DCMU 3-(3,4-chlorophenyl)-1,1-dimethylurea - DPC diphenylcarbazide - DPIP 2,6-dichlorophenylindophenol     

PHYSIOLOGICAL CHANGES DURING GERMINATION OF PITHOPHORA OEDOGONIA (CHLOROPHYCEAE) AKINETES1

Akinetes of Pithophora oedogonia (Mont.) Wittrock, formed in stationary phase cultures, were induced to germinate by transfer to fresh media. Changes in various physiological parameters were monitored during the first 20 d of germination. Ungerminated akinetes contained a greater percentage of lipid, starch, and dry matter and exhibited lower photosynthetic rates than did vegetative cells. Germination consisted of four phases. Phase I (day-0 to day-1) was marked by a rapid increase in respiratory rates. Phase II (day-2 to day-6) involved protrusion of the germination tube and was insensitive to cyanide, a respiratory inhibitor, and simazine, a photosynthetic inhibitor. Phase III (day-7 to day-14) was marked by rapid elongation of the germination tube, increasing photosynthetic rate, increasing chlorophyll and water content and declining levels of lipid and starch. Germination tube elongation during phase III was not inhibited by simazine, but was cyanide sensitive. Phase IV (day-15 to day-20) was characterized by a reduction in respiratory rate and an abrupt increase in the ratio of photosynthesis to respiration. Germination tube elongation during phase IV was inhibited by simazine. The data indicate that germination in Pithophora oedogonia akinetes consists of an extensive period (phases I, II and III) during which reserve materials are respired. Utilization of internal food reserves apparently permits akinetes to germinate and supports the initial growth of the germination tube in light limited microenvironments.     

Environmental influences on akinete germination of Anabaena circinalis and implications for management of cyanobacterial blooms

Certain cyanobacteria, including the noxious bloom-forming species Anabaena circinalis Rabenhorst, produce thick-walled reproductive structures (akinetes) which may serve as a resting stage and ensure survival during adverse growth conditions. The effect of certain environmental variables (temperature, salinity and desiccation) on akinete germination of A. circinalis was investigated under laboratory conditions, to determine the conditions under which germination was inhibited. The overall aims were to provide a broader understanding of the life history and ecology of this species and to assess suppression of akinete germination as a potential management strategy for control of cyanobacterial blooms in the lower Murray River, Australia. The results indicated a marked threshold of temperature and salinity tolerance for germination of A. circinalis, but the latter was not within a range that could be successfully manipulated in a natural ecosystem. However, it was found that desiccation of akinetes for moderately short periods can significantly impair their capacity to germinate. It is, therefore, speculated that allowing periodic drying of shallow wetlands adjacent to the Murray River and in other areas may reduce the size of the inoculum for population growth by reducing viability of akinetes in surface sediments.     

AKINETE GERMINATION IN ANABAENA CIRCINALIS (CYANOPHTA)

Akinetes of a clonal culture of Anabaena circinalis Rabenhorst from Mt. Bold reservoir (eutrophic), South Australia, were isolated and the effects of light, phosphorus, and nitrogen availability on their germination were investigated. Light was required but there was no significant difference in percentage of germination after 72 h if akinetes were incubated in ASM-1 medium at irradiances of 15, 30, or 50 μmol.m^-2.s^-1. Maximum akinete germination occurred by 48 h. Nitrogen was not required, as 88% of akinetes germinated in the flasks without combined nitrogen added to the medium and without N₂ in the air. NH₄⁺-N at 28 mg N.L^-1 completely suppressed germination, whereas 28 mg NO₃ N.L^-1 had no effect relative to the controls without nitrogen. Phosphorus was required, and at 48 h percentage of germination in the flasks with 0.6 mg P.L^-1 added (78%) was significantly greater than in the flasks with 0.06 P.L^-1 (58%) and 0 mgP.L^-1 (24%) added. Germlings in the 0 mg P.L^-1 flasks were only 2–4 cells long and stunted in appearance, whereas germlings at all other P concentrations were 8–16 cells long. It is likely that the isolation process exposed some akinetes to intracellular phosphorus released from lysing vegetative cells, but this was insufficient to allow normal development in the 0 mg P.L^-1 flasks. The plot of percentage of germination vs. initial phosphorus concentration, in the medium showed a relationship analogous to Michaelis-Menten nutrient uptake kinetics, suggesting that a specific membrane-bound enzyme system(s) is involved, with phosphorus as the substrate. The half saturation value (K_S) for germination was 50 μg P.L^-1.     

Metabolic activities of isolated akinetes of the cyanobacterium Nostoc spongiaeforme.

Intact akinetes (spores) of the cyanobacterium Nostoc spongiaeforme can be isolated free of vegetative cells and heterocysts. The akinetes remain viable for at least 2 weeks in distilled water. They do not germinate in water but do so readily when transferred subsequently to cyanobacterial growth medium. Isolated, nongerminating akinetes incorporated 35S from Na235SO4 into protein and lipid. Similar incorporation was observed when akinetes were isolated from old cultures (containing primarily akinetes) which were labeled with Na235SO4 for 4 to 5 h before isolation. The metabolic activities of isolated akinetes were therefore not a factitious response to the isolation procedure. Autoradiographs of radioactive akinetes showed that 35S was incorporated by virtually all akinetes, rather than by a small subpopulation of active cells. Akinetes consumed O2 in the dark and, in a dichlorophenyl dimethylurea-sensitive reaction, evolved O2 in the light. We conclude that akinetes are metabolically active under conditions in which germination does not occur.     

Factors Affecting the Germination of Akinetes of Nodularia spumigena (Cyanobacteriaceae)

Nutritional and physical factors which influence the germination of akinetes of Nodularia spumigena (Cyanobacteriaceae) were examined. Low concentrations of phosphorus (<0.9 μM) were required for germination. Nitrate had no effect, but ammonia, at concentrations of >45 μM, inhibited germination. Salinities of >20‰ were inhibitory to germination. Optimum temperatures were 22°C or greater. Germination did not take place in the dark, but only very low light intensities (0.5 microeinstein m⁻² s⁻¹) were necessary to initiate germination. Red light (620 to 665 nm) was required. More than 24 h of continuous exposure to light was necessary for any significant germination to occur. The conditions for germination corresponded with conditions in the Peel-Harvey Estuary, Western Australia, 2 to 3 weeks before large summer Nodularia blooms.     

Interferometric studies of the dynamics of hydration and dry matter content during light-dependent germination of the Anabaena variabilis Kützing akinetes]

Calculations following interference-microscopical measurements performed on akinetes (A), heterocyts (H), and "vegetative" cells (F) of the Cyanobacterium (blue-green alga) Anabaena variabilis resulted in significant higher values of mean absolute dry matter content of the akinetes (2.06 . 10(-10) g; as compared to 0.46 . 10(-10) g and 0.31 . 10(-10) g for H and F, respectively). tthe water content of these resting cells (63%) was significantly lower than in the other two types of cells (H: 85%, F: 77%). Light exposition of the akinetes in fresh nutrition medium (i. e., conditions allowing germination within 30--50 h) resulted in a decrease of the relative dry matter content so that already in the period preceding the outgrowth of the germling the water content of the vegetative cells was achieved. Simultaneously their volume increased by the uptake of water; whereas the absolute content of dry matter remained constant or was even temporarily diminished during the first period. Only in the second period the values increased in some cases and then remained constant up to germination. The increased dry matter content, however, was not a precondition necessary for the germination of the akinetes. In darkness under otherwise unaltered conditions the values remained unchanged or, after a light period, came back to the initial level. The results demonstrate that formation and germination of the resting cells of Cyanobacteria as well are connected with an alteration in the hydratation level, i. e., in cells which continuously are kept under water saturated conditions. This increase by hydratation during the germination period is, as the germination process itself, strictly controlled by light.     

PHOTOSYNTHETIC CHARACTERIZATION OF DEVELOPING AND MATURE AKINETES OF APHANIZOMENON OVALISPORUM (CYANOPROKARYOTA)1

Akinetes, differentiated resting cells produced by many species of filamentous, heterocystous cyanobacteria, enable the organism to survive adverse conditions, such as cold winters and dry seasons, and to maintain germination capabilities until the onset of suitable conditions for vegetative growth. Mature akinetes maintain a limited level of metabolic activities, including photosynthesis. In the present study, we have characterized changes in the photosynthetic apparatus of vegetative cells and akinetes of the cyanobacterium Aphanizomenon ovalisporum Forti (Nostocales) during their development and maturation. Photosynthetic variable fluorescence was measured by microscope‐PAM (pulse‐amplitude‐modulated) fluorometry, and the fundamental composition of the photosynthetic apparatus was evaluated by fluorescence and immunological techniques. Vegetative cells and akinetes from samples of Aphanizomenon trichomes from akinete‐induced cultures at various ages demonstrated a gradual reduction, with age, in the maximal photosynthetic quantum yield in both cell types. However, the maximal quantum yield of akinetes declined slightly faster than that of their adjacent vegetative cells. Mature akinetes isolated from 6‐ to 8‐week‐old akinete‐induced cultures maintained only residual photosynthetic activity, as indicated by very low values of maximal photosynthetic quantum yields. Based on 77 K fluorescence emission data and immunodetection of PSI and PSII polypeptides, we concluded that the ratio of PSI to PSII reaction centers in mature akinetes is slightly higher than the ratio estimated for exponentially grown vegetative cells. Furthermore, the cellular abundance of these protein complexes substantially increased in akinetes relative to exponentially grown vegetative cells, presumably due to considerable increase in the biovolume of akinetes.     

Viability of dried vegetative cells and the formation and germination of reproductive structures inPithophora œdogonia, Cladophora glomerata andRhizoclonium hieroglyphicum under water stress

All dried vegetative cells ofPithophora œdogonia died within 1 h, while those ofCladophora glomerata andRhizoclonium hieroglyphicum retain viability to some extent for 1 and 8 d, respectively, under similar storage conditions. The viability of dried vegetative cells of eitherC. glomerata orR. hieroglyphicum decreased more or less equally when stored either at 20 °C. in light or dark or at 12 °C in dark, but was lost rapidly and drastically when stored at 0 °C in dark. Both dried and wet akinetes ofP. œdogonia were equally more viable when stored at 20 °C in dark than in light, but they lost germination ability when stored either at 12 or 0 °C in dark; this might be either due to loss of viability or dormancy induction at low temperatures. The water stress imposed by growing vegetative filaments either on highly agarized media, in NaCl-supplemented liquid media or in media undergoing progressive air-drying to complete dryness did not induce, but reduced akinete formation inP. œdogonia, decreased zoosporangium formation inC. glomerata andR. hieroglyphicum, decreased or totally suppressed akinete germination inP. œdogonia and zoospore germination inC. glomerata andR. hieroglyphicum. Akinetes ofP. œdogonia formed under water stress were equally viable, while zoosporangia ofC. glomerata andR. hieroglyphicum formed under water stress were comparatively less viable than those formed without any water stress. Akinete germination inP. œdogonia and zoospore germination inC. glomerata andR. hieroglyphicum were comparatively more sensitive to water stress than the formation of akinetes and zoosporangia. The akinete germination inP. œdogonia was more sensitive to water stress than zoospore germination inC. glomerata andR. hieroglyphicum and it might be either due to their large size, thick wall or dense content.     

Structural aspects of akinete germination in the cyanobacterium Anabaena variabilis

Electronmicroscopical investigations of light activated akinetes in different phases before outgrowth of the germinating cell showed two alterations in the akinete envelope, obviously in connection with the germination process. After induction of germination the akinetes show formation of an expanding more or less electron dense layer between the outer cell wall layer (outer membrane, L_IV) and the condensed part of the akinete coat (the transformed sheath of the vegetative cell). Between this new formed layer and the mentioned part of the akinete coat thick laminar layers are deposited which contain alternately electron dense and electron transparent strata. The expanding layer is assumed to be a mucous layer which acts as swelling body causing, after bursting of the layered shell, the expulsion of the germinating cell in the manner characteristic for Anabaena variabilis.     

Akinetes of the cyanobacteriumNostoc PCC 7524: morphological changes during synchronous germination

Following dilution into fresh medium in the light, akinetes ofNostoc PCC 7524 germinated synchronously. Synchrony was maintained at a high level during the first 24 h, at which time the young filaments were composed either of three cells (with N₂ as nitrogen source) or four cells (with NO ₃ ^- or NH ₄ ⁺ ), and at a slightly lower level during the next 24 h of growth. The pattern of cell division was similar in media containing the different nitrogen sources although the timing of the major events varied. In the presence of N₂ or NO ₃ ^- , heterocysts differentiated synchronously; the first developed invariably from a terminal cell of the young filament at approximately 19 h, the second from the other terminal cell after further vegetative cell division. Heterocyst differentiation did not occur in the presence of NH ₄ ⁺ . In the absence of nitrogen (gas phase argon: CO₂) akinete germination initially followed the same pattern as that observed in N₂, this early stage probably occurring at the expense of intracellular reserve materials.During germination, a new laminated layer, similar in structure and position to that found in the heterocyst envelope, appeared in the akinete envelope. This layer was not present in the germinating akinetes of a mutant which was incapable of forming heterocysts.     

Influence of Light on Germination of Pinus palustris Seeds

Red light with a wavelength of 660 nm promotes germination of longleaf pine (Pinus palustris Mill.) seeds, and far-red light (730 nm) inhibits germination. The promotion-inhibition process is repeatedly reversible, indicating that germination is controlled by the photoreversible reaction of phytochrome. Response varied greatly between single-tree lots and was dependent on the length of time seeds were imbibed at 5°C. Dry seeds did not respond to light treatments when they were subsequently imbibed and tested in darkness. Stratification for 28 days essentially removed the light requirements for germination.