VEGETATIVE GROWTH OF ACETABULARIA ACETABULUM (CHLOROPHYTA): STRUCTURAL EVIDENCE FOR JUVENILE AND ADULT PHASES IN DEVELOPMENT1

We characterized vegetative development in two inbred cell lines of Acetabularia acetabulum (L.) Silva. Cell growth occurred at the apex and by elongation of older interwhorls throughout vegetative development. Although cell length and hairs per whorl increased regularly during development, interwhorl length, hair persistence on the stalk, and complexity of each whorl (degree of branching of whorl hairs) showed sharp discontinuities during development in both cell lines. The first (earliest) discontinuity, formation of a short interwhorl, was the sixth interwhorl made in all cells. Even though cell line Aa1055 was twice the height ofAa4010 when mature, cells in both lines were 0.8–1.0 cm tall after formation of the short interwhorl. The second discontinuity, increases in hair persistence on the stalk and complexity of each whorl of hairs, began shortly before cap initiation. We propose the following nomenclature: 1) that slower growth before formation of the short interwhorl be called “juvenile”; 2) that more rapid growth after formation of the short interwhorl be called “adult”; and 3) that adult growth be separated into “early” and “late” phases by the discontinuities in whorl hair characteristics. The proposed developmental phases (juvenile, early adult, and late adult) are temporally sequential and spatially stacked.     

EFFECTS OF NITROGEN SOURCES ON P-LIMITED GROWTH OF ANABAENA FLOS-AQUAE1

The effects of N₂, nitrate and ammonia as N sources were investigated in P-limited and nutrient-sufficient cultures of Anabaena flos-aquae (Lyngb.) Bréb. The maximum growth rate (μ_m) was highest at 1.34 d^?1 with ammonia, compared to 1.18 with nitrate and 0.95 d^?1 with N₂. There was no difference in P requirement between N₂ and nitrate cultures. Under P-limited conditions, the increase in cell P with growth rate (μ) was identical. With N₂ as the N source, cell-N concentrations in P-limited cells increased with μ as did cell P, and the cellular N:P ratio remained the same (14) within the range of μ examined. With nitrate, however, cell N concentrations were high and independent of n, except at a low μ. It appears that this organism fixes atmospheric N₂ only at the minimum concentration required to maintain a μ. The acetylene reduction rate increased with μ in both N_2- and nitrate-grown cells, but the rate was lower in nitrate. Under P-limitation, there was no difference in net C-fixation rate per cell between N₂ and nitrate cultures at a given μ. However, the rate per unit of chlorophyll a (chl a) was higher in N₂ than in nitrate cultures, and the rate was independent of μ with N₂ but was a linear function of nitrate supplied. The maximum C-fixation rate in nutrient sufficient cells was highest with ammonia, followed by nitrate and N₂. The cellular chl a concentration was correlated with the total cell-N concentrations regardless of H and the source of N.     

BRIEF INCUBATION OF GAMETANGIA-BEARING CAPS IN ANTIBIOTICS ELIMINATES BRANCHING IN PROGENY OF ACETABULARIA ACETABULUM (CHLOROPHYTA)1

Branching of the stalk of Acetabularia acetabulum L. (Silva) was investigated by inbreeding and by a brief treatment of gametangia with a variety of antibiotics. The position of the branch along the stalk varied, implying that branching was not restricted to any one time in development (base is oldest and apex is youngest). The branching phenotype was not inherited in Mendelian fashion. Although three microscopic structures (“bubbles,”“pustules,” and “scars”) occurred on the stalks of cells that had branched, these structures were not statistically correlated with branching in the population (n=699 cells). However, brief treatment of gametangia with a new antibiotic mixture did eliminate all macro- and microscopic structures associated with branching of the stalk in the subsequent generation. We could not fulfill Koch's postulates or provide clear evidence for the pathogenic nature of cell branching. Our brief antibiotic treatment of gametangaa of Acetabularia acetabulum was rapid, had no adverse effects, and virtually eliminated branching (and any potential pathogens) from laboratory cultures in the subsequent generations. Our method allows biochemical and molecular analyses to proceed uncomplicated by the possible presence of other organisms and provides a clean baseline for the future selection of mutations that may induce heritable branching.     

氮磷水平对龙须菜生长和光合特性的影响

研究不同营养盐条件对龙须菜(Gracilaria lemaneiformis)的生理效应, 对深入了解龙须菜与近海环境的相互作用具有重要意义。在低氮低磷(LNLP)、低氮高磷(LNHP)、高氮低磷(HNLP)和高氮高磷(HNHP) 4种营养盐条件下培养龙须菜15 d, 以探讨不同氮、磷水平对龙须菜生长和光合特性的影响。结果表明: 1) LNHP、HNLP和HNHP处理促进了龙须菜的生长, 其中HNHP处理下龙须菜具有最大的相对生长速率和生物量; 2) LNHP、HNLP和HNHP处理提高了龙须菜的光合无机碳利用能力, 其中HNHP处理下龙须菜具有最大的无机碳饱和光合速率和表观半饱和常数, 比LNLP处理分别提高了118%倍和48.71%; 3) LNHP、HNLP和HNHP处理显著影响龙须菜的光化学效率, 与LNLP处理相比, LNHP处理提高了龙须菜的光化学效率, 而HNLP和HNHP处理降低了龙须菜的光化学效率。研究结果表明, HNHP处理条件下, 龙须菜的生长和光合无机碳利用能力最高, 光化学效率最低。     

PHYSIOLOGICAL FACTORS THAT AID DIFFERENTIATION OF ZYGOTES AND EARLY JUVENILES OF ACETABULARIA ACETABULUM (CHLOROPHYTA)

To improve our understanding of early development and to enable standard genetic analyses in Acetabularia acetabulum (L.) Silva, we examined some physiological factors that impact differentiation of young diploid plants. Eighty percent of zygotes differentiated within 6–7 days when they were inoculated at high densities (≥5,000 zygotes·mL⁻¹), and zygote differentiation was independent of the photon flux density of cool white light. In contrast, 90% of juveniles differentiated within 25 days when they were inoculated at medium densities (<1,000 individuals·mL⁻¹) and required >250 μmol·m⁻²·s⁻¹ cool white light. Both zygotes and juveniles differentiated best at an alkaline pH (7.96), when the temperature was between 18.6° and 23.5° C, and when the external potassium concentration was 1–10 mM. Compared to prior publications, adjustment of these parameters reduced the time to differentiation by 25% (from 8 to 6 days) and more than tripled the percentage of zygotes that developed into healthy juveniles (from 23% to ∼80%).     

MULTIMINERAL CALCAREOUS DEPOSITS IN THE MARINE ALGA ACETABULARIA ACETABULUM (CHLOROPHYTA; DASYCLADACEAE)

The ultrastructure and mineralogy of cultured and wild specimens of the green algae Acetabularia acetabulum were studied. Sites of calcification were clarified using TEM and SEM. Minerology was determined via electron diffraction, energy-dispersive x-ray microanalysis, and Fourier transform infrared spectral analyses of the calcareous deposits. These deposits were found covering the surface and within the cell wall of the cultured juveniles and adult wild specimens of A. acetabulum. Deposits within and directly over the wall were amorphous calcium carbonate (ACC) in granular form. In-wall and outer cell wall ACC appear to develop independently from each other. The formation of the in-wall ACC may be mediated by the wall structure and/or chemistry, whereas mucin may be the factor mediating formation of outer cell wall ACC. In contrast to the stable in-wall ACC, outer cell wall ACC could be transitory, transforming into aragonite. In cultured cells, a small amount of monohydrocalcite crystals and calcite were found. It is possible that these were transformation products of ACC. In wild specimens, whewellite (calcium oxalate monohydrate) was present inside and outside of the cap wall. It is unclear if whewellite is present within the cytoplasm. Local and temporal differences in the microenvironments for the multiphase mineralization are suggested.     

A NEW,ARTIFICIAL SEA WATER THAT FACILITATES GROWTH OF LARGE NUMBERS OF CELLS OF ACETABULARIA ACETABULUM (CHLOROPHYTA) AND REDUCES THE LABOR INHERENT IN CELL CULTURE1

We designed a new, artificial seawater (Ace 25) in order to grow bulk cultures of Acetabularia acetabulum (L.) Silva with a minimum of labor. To this end, we modified the traditional recipe for cell growth (Müller's medium as modified by Schweiger et al.) We eliminated five of the inorganic chemicals and determined the optimum concentration for 16 of the remaining 18 inorganic chemicals from modified Müller's seawater. Ace 25 enables growth of A. acetabulum from the beginning of the juvenile phase through gametangial formation in 11 weeks at high cell densities without medium replenishment. This represents a 98% reduction in the seawater volume required to mature each cell, a 30–40% reduction of the duration of the life cycle, an estimated 80% reduction in labor, and a 50–95% reduction in the space required for culturing A. acetabulum as compared with traditional procedures. These improvements may facilitate studies that require large numbers of cells such as population studies, genetics, and biochemistry, contribute to understanding the nutritional requirements of marine algae, and extend the use of this cell to those who lack the space or manpower to grow the cells in the traditional manner.     

PARTICIPATION OF EXTRACELLULAR NUCLEOTIDES IN THE WOUND RESPONSE OF DASYCLADUS VERMICULARIS AND ACETABULARIA ACETABULUM (DASYCLADALES,CHLOROPHYTA)1

As assayed by fluorescent reporter dyes, nitric oxide (NO) and H₂O₂, two downstream signaling agents induced by wounding in the alga Dasycladus vermicularis (Scop.) Krasser, can also be induced in unwounded Dasycladus cells by μM Adenosine 5′[γ‐thio]triphosphate (ATPγS) and Adenosine 5′‐[β‐thio]diphosphate (ADPβS), but not by Adenosine 5′‐O‐thiomonophosphate (AMPS). These nucleotide‐induced responses are blocked by pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), an antagonist of animal purinoceptors, and by adenosine, a feed‐back inhibitor of extracellular nucleotide responses in animals. Similar nucleotide‐ and nucleotide‐antagonist responses were observed in Acetabularia acetabulum (L.) P. C. Silva. Significant levels of ATP released from Dasycladus cells were measured at wound sites by a sensitive luciferin‐luciferase assay. Additionally, the normal wound‐induced production of NO and H₂O₂ in Dasycladus can be blocked by pretreating the cells with PPADS. Our results indicate that nucleotides released from wounds can serve as a signal to trigger wound responses in algae, and that coordinated signaling between extracellular nucleotides and the NO pathway may have been established early during the evolution of plants.     

不同氮源及CO_2浓度下湖泊微拟球藻的生长及产油特性分析

<正>工业生产排放的各种废气与污水污染人类赖以生存的环境。化石燃料燃烧除排放出大量CO2外,还释放出含有粉尘、SO[x和NOx等直接危害人类健康的有毒成分1]。其中NO]x与水结合后最终会转化成硝酸盐或亚硝酸盐等[2,导致污水中的含氮化合物氨氮、亚硝酸盐氮、硝酸盐氮和有机氮的含量通常偏高。如何有效去除污水中的氮是防治水体污染最关键的步骤之一,而利用微藻培养去除水体中的氮源是目前研究的热点。尽管生物燃料生产的成本远     

CHANGES IN INTRACELLULAR NITROGEN POOLS AND FEEDBACK CONTROLS ON NITROGEN UPTAKE IN CHAETOMORPHA LINUM (CHLOROPHYTA)1

Changes in the size of intracellular nitrogen pools and the potential feedback by these pools on maximum N uptake (NH₄⁺ and NO₃^?) rates were determined for Chaetomorpha linum (Müller) Kützing grown sequentially under nutrient-saturating and nutrient-limiting conditions. The size of individual pools in N-sufficient algae could be ranked as residual organic N (RON) comprised mainly of amino acids and amino compounds > protein N > NO₃^? > NH₄⁺ > chlorophyll N. When the external N supply was removed, growth rates remained high and individual N pools were depleted at exponential rates that reflected both dilution of existing pools by the addition of new biomass from growth and movement between the pools. Calculated fluxes between the tissue N pools showed that the protein pool increased throughout the N depletion period and thus did not serve a storage function. RON was the largest storage reserve; nitrate was the second largest, but more temporary, storage pool that was depleted within 10 days. Upon N resupply, the RON pool increased 3 × faster than either the inorganic or protein pools, suggesting that protein synthesis was the rate-limiting step in N assimilation and caused a buildup of intermediate storage compounds. Maximum uptake rates for both NH₄⁺ and NO₃^? varied inversely with macroalgal N status and appeared to be controlled by changes in small intracellular N pools. Uptake of NO₃^? showed an initial lag phase, but the initial uptake of NH₄⁺ was enhanced and was present only when the intracellular NH₄⁺ pool was depleted in the absence of an external N supply. A strong negative correlation between the RON pool size and maximum assimilation uptake rates for both NH₄⁺ and NO₃^? suggested a feedback control on assimilation uptake by the buildup and depletion of organic compounds. Enhanced uptake and the accumulation of N as simple organic compounds or nitrate both provide a temporary mechanism to buffer against the asynchrony of N supply and demand in C. linum.     

THE EFFECT OF GROWTH IRRADIANCE ON THE COUPLING OF CARBON AND NITROGEN METABOLISM IN CHAETOMORPHA LINUM (CHLOROPHYTA)

The influence of growth irradiance on the non-steady-state relationship between photosynthesis and tissue carbon (C) and nitrogen (N) pools in Chaetomorpha linum (Muller) Kutzing in response to abrupt changes in external nitrogen (N) availability was determined in laboratory experiments. For a given thallus N content, algae acclimated to low irradiance consistently had a higher rate of light-saturated photosynthesis (P_max normalized to dry weight) than algae acclimated to saturating irradiance; for both treatments, P_max was correlated to thallus N. Both P_max and the photosynthetic efficiency (α_dw) were correlated in C. linum grown at either saturating or limiting irradiance over the range of experimental conditions, indicating that variations in electron transport were coupled to variations in C-fixation capacity despite the large range of tissue N content from 1.1% to 4.8%. Optimizing both α and P_max and thereby acclimating to an intermediate light level may be a general characteristic of thin-structured opportunistic algae that confers a competitive advantage in estuarine environments in which both light and nutrient conditions are highly variable. Nitrogen-saturated algae had the same photosynthesis–irradiance relationship regardless of light level. When deprived of an external N supply, photosynthetic rates did not change in C. linum acclimated to low irradiance despite a two-fold decrease in tissue N content, suggesting that the active pools of chlorophyll and Rubisco remained constant. Both α and P_max decreased immediately and continuously in algae acclimated to high irradiance on removal of the N supply even though tissue N content was relatively high during most of the N-starvation period, indicating a diversion of energy and reductant away from C fixation to support high growth rates. Carbon and nitrogen assimilation were equally balanced in algae in both light treatments throughout the N-saturation and -depletion phases, except when protein synthesis was limited by the depletion of internal N reserves in severely N-starved high-light algae and excess C accumulated as starch stores. This suggests that the ability for short-term adjustment of internal allocation to acquire N andC in almost constant proportions may be especially beneficial to macroalgae living in environments characterized by high variability in light levels and nutrient supply.     

TIMING AND LIGHT REGULATION OF APICAL MORPHOGENESIS DURING REPRODUCTIVE DEVELOPMENT IN WILD-TYPE POPULATIONS OF ACETABULARIA ACETABULUM (CHLOROPHYCEAE)

In the giant unicellular green alga, Acetabularia acetabulum (L.) Silva, development is altered by light. For example, blue light induces the vegetative apex to produce whorls of hairs that encircle the stalk and, later, blue light may trigger reproductive onset. The two goals of this study were to determine when changes in apical shape occur during formation of the reproductive structure, or "cap," and to determine which of these differentiation events require light. The first visible indication of cap initiation was a rounded swelling of the apex, which we call a knob-shaped apex (time = 0 hours). Subsequent changes in shape were a hyaline, knob-shaped apex, reached by 50% of the population 3 h later, and the formation of a whorl of unilobed chambers at 16 h. These chambers became bilobed at 33 h and trilobed at 34 h. Successive sets of cap hairs grew from protuberances found on the surface of the uppermost lobes of the chambers (superior corona). After knob, the remainder of cap formation was largely independent of light. However, the initiation of each set of cap hairs required light. If a recently initiated cap was amputated, the individual recapitulated development, repeating a portion of vegetative morphogenesis (i.e. it made whorls of sterile hairs) before initiating a new cap. The developmental sequence between amputation and initiation of a new cap required light. A model for light-regulated changes in shape at the apex of Acetabularia acetabulum, which integrates whorl and cap formation and encompasses both vegetative and reproductive development of this organism, is presented.     

EFFECTS OF EXTERNAL INORGANIC NITROGEN CONCENTRATION ON METABOLISM,GROWTH AND ACTIVITIES OF KEY CARBON AND NITROGEN ASSIMILATORY ENZYMES OF LAMINARIA SACCHARINA (PHAEOPHYCEAE) IN CULTURE1

The growth rate of Laminaria saccharina (L.) Lamour. is dependent on inorganic nitrogen in culture. Growth rates were saturated between 5 and 10 μmol · L^?1 nitrate. The activities of ribulose-1,5 bisphosphate carboxylase, phosphoenolpyruvate carboxykinase, mannitol-1-phosphate dehydrogenase, nitrate reductase and glutamine synthetase also varied with the concentration of inorganic nitrogen in the medium. All enzyme activities were lowest at 2.5 μmol · L^?1 nitrate (the lowest concentration used) increasing to a maximum activity between 10 and 30 μmol · L^?1 nitrate. Most enzyme activities followed a hyperbolic curve resembling those described by the Michaelis-Menten equation, with different half-saturation constants.     

EFFECT OF NITROGEN AND PHOSPHORUS SUPPLY ON GROWTH AND TISSUE COMPOSITION OF ULVA FENESTRATA AND ENTEROMORPHA INTESTINALIS (ULVALES,CHLOROPHYTA)1

The chlorophyte macroalgae Ulva fenestrata (Postels and Ruprecht) and Enteromorpha intestinalis (Linnaeus) Link. were grown under various nutrient regimes in indoor semi-continuous and batch cultures. Tissue nitrogen contents ranged from 1.3–5.4% N (dry wt), whereas tissue P ranged from 0.21–0.56% P (dry wt). Growth in low nitrogen medium resulted in N:P ratios of 5–8, whereas growth in high nitrogen medium resulted in N:P ratios of 21–44. For U. fenestrata, tissue N:P < 16 was indicative of N-limitation. Tissue N:P 16–24 was optimal for growth and tissue N:P > 24 was indicative of P-limitation. Growth of U. fenestrata was hyperbolically related to tissue N but linearly related to tissue P. Phosphorus-limited U. fenestrata maintained high levels of tissue N, but N-limited algae became depleted of P. For E. intestinalis, tissue N remained at maximum levels during P-limitation whereas tissue P decreased to about 85% of maximal levels during N-limitation. Growth rates for U. fenestrata decreased faster during P-limitation than during N-limitation. Simultaneously, tissue P was depleted faster than tissue N. Our results suggest that comparing tissue N and P of macroalage grown in batch cultures is useful for monitoring the nutritional status of macroalgae.     

AXENIC CULTURES OF ACETABULARIA (CHLOROPHYTA): A DECONTAMINATION PROTOCOL WITH POTENTIAL APPLICATION TO OTHER ALGAE1

We developed an easy, reliable decontamination protocol for caps of Acetabularia acetabulum (L.) Silva; with minimal labor hundreds of caps can be decontaminated. In addition, cysts isolated from these caps do not exhibit dormancy and can be used immediately to establish large populations of axenic cell cultures. This method consists of three successive incubations: 1) proteinase K/sodium dodecyl sulfate/1,3-bis[tris(hydroxymethyl)-methylamino]propane for 1 h, 2) mild silver protein for 5 min, and 3) an antibiotic solution (neomycin, chloramphenicol, nystatin, ampicillin, streptomycin)for 3 days. This protocol eliminates bacterial, algal, fungal, and yeast contaminants. It is useful for decontaminating caps from lab cultures and caps collected from the wild and may also be effective in decontaminating the reproductive structures of other algae. Cyst dormancy was reduced from 15 weeks to less than 1 week, which represents a 40% reduction of the life cycle of A. acetabulum. We routinely obtained 90-100% gamete release from cysts 3-14 days after they were made axenic. The component of our culturing methods that allows gamete release without a “dormant” period is unknown.     

NITROGEN LIMITATION EFFECTS OF DIFFERENT NITROGEN SOURCES ON NUTRITIONAL QUALITY OF TWO FRESHWATER ORGANISMS, SCENEDESMUS QUADRICAUDA (CHLOROPHYCEAE) AND SYNECHOCOCCUS SP. (CYANOPHYCEAE)

Food quality for grazers has been related to mineral (nitrogen, phosphorus) and biochemical (amino acids, fatty acids) constituents. The aim of the study was to examine the influence of different nitrogen sources on these constituents in two organisms, the green alga Scenedesmus quadricauda Turp. and the cyanobacterium Synechococcus sp., commonly used in feeding experiments. The two organisms were grown in continuous cultures at different growth rates. Nitrate or ammonium salts were used as nitrogen sources under both replete and limited conditions. Carbon content (mg·g⁻¹ dry weight) was stable in both organisms independent of nitrogen source, nitrogen limitation, and growth rate. Nitrogen content decreased with limitation and growth rate in Scenedesmus and to a lesser degree in Synechococcus , whereas changes in phosphorus content were not statistically significant. The relative proportions of amino acids (% of total amino acids) were relatively stable in both organisms, whereas the proportions of fatty acids varied with growth rate and limitation. Fatty acid content was much lower in Synechococcus than in Scenedesmus . At N limitation, polyunsaturated fatty acids (PUFAs) showed lower levels in both organisms. The change occurred in the ω3 PUFA (linolenic acid) of the green alga and in the ω6 PUFA (linoleic acid) of the cyanobacterium. The difference in the response of N limitation in the two organisms may be traced to the different composition of the chloroplast membranes (the prokaryotic way) and the microsomal membranes (the eukaryotic way) where the desaturation takes place.     

EFFECTS OF SELECTED PHYSICOCHEMICAL FACTORS ON GROWTH AND ZOOSPOROGENESIS OF CLADOPHORA GLOMERATA (CHLOROPHYTA)1

The effects of vitamin B₁ and B₁₂, temperature, light intensity and photoperiod on dry weight production and zoosporogenesis of Cladophora glomerata (L.)Kütz. were investigated by factorial experiments. Statistical analysis showed all of the above factors to be significantly (P < 0.01) influenced by these two parameters. Zoosporogenesis appeared to be more sensitive to vitamin limitation than dry weight production. Dry weight production and zoosporogenesis exhibited significantly different maxima. Dry weight production was most strongly influenced by temperature whereas photoperiod exerted the strongest influence on zoosporogenesis. It was possible to experimentally separate the light intensity and photoperiod factors; analysis showed photoperiod to be the primary factor influencing zoosporogenesis. Among the conditions tested, an 8:16 h LD short day photoperiod elicited the greatest number of zoosporangia.     

氮源及其浓度对三角褐指藻生长和脂肪酸组成的影响

为了研究氮源的类型和浓度对微藻脂肪酸组成的影响 ,用含有不同浓度NO3 -、NH4 、NH2 CONH2 的培养基 ,对三角褐指藻 (Phaeodactylumtricornutum)进行了培养 ,并测定了其生长和脂肪酸组成。结果表明 ,培养基中不添加氮源时 ,三角褐指藻生长缓慢 ,但多不饱和脂肪酸 (PUFAs)和C18脂肪酸 (C18∶0 ,C18∶2 (n -6) ,C18∶3 (n -6) )占总脂肪酸的比例较高 ;氮浓度较低 (<1 8mmol/L)时 ,三角褐指藻以NH4 为氮源 ,生长较快 ;氮浓度较高 (>3 5mmol/L)时 ,以NH2 CONH2 为氮源 ,生长较快。以NH4 或NH2 CONH2 为氮源时 ,EPA(Eicosapentaenoicacid)和PUFAs占总脂肪酸的比例随着其浓度的增加而上升 ;而以NO3 -为氮源时 ,EPA和PUFAs随着NO3 -浓度增加先上升后下降 ,最适NO3 -浓度为 1 8mmol/L ,此时的EPA占总脂肪酸的比例为 16 7%。EPA占干重 (w/w)的比例 ,不管是哪种氮源 ,均随着氮浓度的增加而升高 ,但是在 0 9— 3 5mmol/L之间 ,3种氮源间EPA含量差异不显著。当氮源浓度为 7 0mmol/L时 ,以NH2 CONH2 为氮源 ,EPA和PUFAs含量最高 ,分别为 2 6 %和 4 4 %。PUFAs占干重的比例随着NO3 -浓度增加而下降 ,随NH2 CONH2 浓度增加而升高 ,而受NH4 浓度变化的影响不显著。