Minicircular plastid DNA in the dinoflagellate Amphidinium operculatum

Plastid DNA was purified from the dinoflagellate Amphidinium operculatum. The genes atpB, petD, psaA, psbA and psbB have been shown to reside on single-gene minicircles of a uniform size of 2.3–2.4 kb. The psaA and psbB genes lack conventional initiation codons in the expected positions, and may use GTA for translation initiation. There are marked biases in codon preference. The predicted PsbA protein lacks the C-terminal extension which is present in all other photosynthetic organisms except Euglena gracilis, and there are other anomalies elsewhere in the predicted amino acid sequences. The non-coding regions of the minicircles contain a “core” region which includes a number of stretches that are highly conserved across all minicircles and modular regions that are conserved within subsets of the minicircles. Received: 8 September 1999 / Accepted: 10 November 1999     

The invariance of macromolecular composition with altered light limited growth rate of Amphidinium carteri (dinophyceae)

The effect of irradiance on the growth rate, macromolecular composition and photosynthetic carbon metabolism of Amphidinium carteri was studied in batch culture. Growth rate increased linearly with increasing irradiance up to a maximum growth rate of 0.04 h^-1 at an irradiance of 80 Em^-2s^-1. In contrast to a number of other studies on both prokaryotic and eukaryotic microorganisms, ours showed that cellular content of RNA, DNA, protein and carbohydrate of A. carteri were invariant with growth rate over the range =0.04 to 0.007 h^-1. The invariant macromolecular composition was correlated with a constant modal cell volume. Chlorophyll and lipid per cell increased with decreasing irradiance. The distribution of [¹⁴C]-bicarbonate in the major end products of photosynthesis after incubation with isotope for 14% of a doubling time showed that the percentage carbon in the chloroform (lipids and pigments) fraction increased with decreasing irradiance while that of the trichloroacetic acid soluble (carbohydrate) fractions decreased. The percentage of isotope in the trichloroacetic acid insoluble (protein) fraction and methanol: water fraction (metabolites) remained constant. Because this species, under light-limited growth, differs from other organisms so far studied, more species must be similarly examined before nucleic acid content is used as an index growth rate in the field.This paper is presented with our best wishes on the occasion of Professor G. Drews 60th birthday     

Subcellular localization of minicircle DNA in the dinoflagellate Amphidinium massartii

Peridinin‐containing dinoflagellates have small circular DNA molecules called minicircle DNAs, each of which encodes one, or occasionally a few, plastid proteins or ribosomal RNA. Dinoflagellate minicircle DNA is composed of two parts: a gene‐coding sequence and a non‐coding sequence that consists of several variable and core regions. The core regions are identical among the minicircle DNAs with different genes within a species or strain. Because such structure is very different from those of well known plastid DNAs, many functional and evolutionary questions have been raised for the minicircle DNAs, and several studies that focus on answering those questions are underway. However, the localization of minicircle DNA is still controversial: several lines of indirect evidence have implied plastid localization, whereas the nuclear localization of minicircle DNA has also been suggested in a species. In order to understand the evolution and function of minicircle DNA, it is important to know its precise localization. In this study, we sequenced two typical minicircle DNAs, one encodes psbA and the other encodes 23S rRNA genes, from an Amphidinium massartii strain (TM16). To determine the subcellular localization of these minicircle DNAs, we performed DNA‐targeted whole cell fluorescence in situ hybridization with A. massartii minicircle DNA‐specific probes and demonstrated that minicircle DNAs were present in plastids. This study provides the first direct evidence for the plastid localization of dinoflagellate minicircle DNAs.     

Influence of long-term limitation of cell proliferation on the cell cycle duration

The hypothesis was tested that constant, long-term inhibition of cell proliferation is the primary cause of cellular senescence. Mouse L-929 fibroblasts were maintained in confluent cultures for periods of 6 and 12 months with cell viability maintained by regular replacement of medium and serum. The mitotic activity of the cell population under these conditions was one mitosis per 10⁴ cells. No changes in the duration of the cell cycle were observed when, following long-term quiescence, the cell were replated and grown at low cell density. The results do not support the hypothesis that prolonged suppression of cell proliferation induces cell senescence.     

Evidence for cAMP-dependent protein kinase in the dinoflagellate,Amphidinium operculatum

A cAMP dependent protein kinase (PKA) was identified in the dinoflagellate Amphidinium operculum. In vitro kinase activity towards kemptide, a PKA-specific substrate, was not detectable in crude lysates. However, fractionation of dinoflagellate extracts by gel filtration chromatography showed PKA-like activity toward kemptide at approximately 66 kDa. These findings suggest that possible low molecular mass inhibitors in crude lysates were removed by the gel filtration chromatography. Pre-incubation of extracts with cAMP prior to chromatography resulted in an apparent molecular mass shift in the in vitro kinase assay to 40 kDa. An in-gel kinase assay reflected activity of the free catalytic subunit at approximately 40 kDa. Furthermore, western blotting with an antibody to the human PKA catalytic subunit confirmed a catalytic subunit with a mass of approximately 40 kDa. Results from this study indicate that the PKA in A. operculatum has a catalytic subunit of similar size to that in higher eukaryotes, but with a holoenzyme of a size suggesting a dimeric, rather than tetrameric structure.     

Effects of ocean acidification and phosphate limitation on physiology and toxicity of the dinoflagellate Karenia mikimotoi

This work demonstrated a 10-day batch culture experiment to test the physiology and toxicity of harmful dinoflagellate Karenia mikimotoi in response to ocean acidification (OA) under two different phosphate concentrations. Cells were previously acclimated in OA (pH = 7.8 and CO₂ = 1100 μatm) condition for about three months before testing the responses of K. mikimotoi cells to a two-factorial combinations experimentation. This work measured the variation in physiological parameters (growth, rETR) and toxicity (hemolytic activity and its toxicity to zebrafish embryos) in four treatments, representing two factorial combinations of CO₂ (450 and 1100 μatm) and phosphate concentration (37.75 and 4.67 umol l⁻¹). Results: OA stimulated the faster growth, and the highest rETR_max in high phosphate (HP) treatment, low phosphate (LP) and a combination of high CO₂ and low phosphate (HC*LP) inhibited the growth and E_k in comparison to low CO₂*high phosphate (LCHP) treatment. The embryotoxicity of K. mikimotoi cells enhanced in all high CO₂ (HC) conditions irrespective of phosphate concentration, but the EC₅₀ of hemolytic activity increased in all high CO₂ (HC) and low phosphate (LP) treatments in comparison of LCHP. Ocean acidification (high CO₂ and lower pH) was probably the main factor that affected the rETR_max, hemolytic activity and embryotoxicity, but low phosphate was the main factor that affected the growth, α, and E_k. There were significant interactive effects of OA and low phosphate (LP) on growth, rETR_max, and hemolytic activity, but there were no significant effects on α, E_k, and embryotoxicity. If these results are extrapolated to the aquatic environment, it can be hypothesized that the K. mikimotoi cells were impacted significantly by future changing ocean (e.g., ocean acidification and nutrient stoichiometry).     

Mortality in cultures of the dinoflagellate Amphidinium carterae during culture senescence and darkness

The study of cell death in higher plants and animals has revealed the existence of an active ('programmed') process in most types of cell, and similarities in cell death between plants, animals, yeast and bacteria suggest an evolutionarily ancient origin of programmed cell death (PCD). Despite their global importance in primary production, information on algal cell death is limited. Algal cell death could have similarities with metazoan cell death. One morphotype of metazoan PCD, apoptosis, can be induced by light deprivation in the unicellular chlorophyte Dunaliella tertiolecta. The situation in other algal taxa is less clear. We used a model dinoflagellate (Amphidinium carterae) to test whether mortality during darkness and culture senescence showed apoptotic characteristics. Using transmission electron microscopy, fluorescent biomarkers, chlorophyll fluorescence and particulate carbon analysis we analysed the process of cell mortality and found that light deprivation caused mass mortality. By contrast, fewer dead cells (5-20% of the population) were found in late-phase cultures, while a similar degenerate cell morphology (shrunken, chlorotic) was observed. On morphological grounds, our observations suggest that the apoptotic cell death described in D. tertiolecta does not occur in A. carterae. Greater similarity was found with paraptosis, a recently proposed alternative morphotype of PCD. A paraptotic conclusion is supported by inconclusive DNA fragmentation results. We emphasize the care that must be taken in transferring fundamental paradigms between phylogenetically diverse cell types and we argue for a greater consistency in the burden of proof needed to assign causality to cell death processes.     

Effects of phosphorous, nitrogen, and carbon limitation on biomass composition in batch and continuous flow cultures of the heterotrophic dinoflagellate Crypthecodinium cohnii

We have used phosphate, nitrogen, or carbon limited batch and continuous flow cultures to study how growth and biochemical composition of the dinoflagellate Crypthecodinium cohnii CCMP 316 is affected by nutrient limitation. Specific contents of phosphorous, proteins, and starch were differently affected by nutrient limitation. The specific phosphorous content in C. cohnii varied 10-20 times depending on phosphate availability in the medium. When phosphate was available it was taken up in excess and stored to be re-utilized during phosphate limitation. The specific protein content varied twofold. At most conditions, proteins made up 12-15% of the biomass dry weight but when cells were nitrogen limited, the specific protein content was only half this value. Floridean starch was the major cell constituent of C. cohnii accounting for 40-50% of the biomass dry weight. Only during carbon limitation did the specific starch content decrease. In contrast was the specific lipid content almost unaffected by nutrient availability and lipids accounted for 12-15% of the biomass dry weight irrespectively of which nutrient that was limiting. Lipid production does therefore not depend on nutrient limitation in C. cohnii and lipids are produced even by carbon limited cells. Cultures grown under phosphate limitation resulted in formation of cells with maximal specific contents of all the three major cell constituents; starch, lipid, and protein.     

Effects of salinity, light and inorganic nitrogen on growth and toxigenicity of the marine dinoflagellate Alexandrium tamarense from northeastern Canada

Growth and toxin production of a highly toxic clone of the marine dinoflagellate Alexandrium tamarense, isolated from the lower St Lawrence estuary (Quebec) in eastern Canada, were studied in unialgal batch cultures under different conditions. Controlled experiments were conducted on the production of paralytic shellfish poisoning (PSP) toxins under conditions of varying light (40, 60, 150, 230 and 470 mol m^-21 s^-1), salinity (10, 15, 20, 25 and 30) and nitrate concentrations (0, 88, 364, 528 and 880 mol l^-1). The effects of variable environmental factors on both toxin composition (% molar) and cell toxicity [pg STXeq (saxitoxin equivalents) cell^-1] were determined through the culture cycle. The toxin profile (% molar; mean SD), determined by high-performance liquid chromatography with fluorescence detection (HPLC-FD), remained stable and was consistently dominated by the low-potency N-sulfocarbamoyl toxins C1/C2 (64.0 ± 3%). There were also substantial relative amounts of the high-toxicity carbamate derivatives gonyautoxin 1-4 (GTX1-4) (1.7 ± 0.5%), neosaxitoxin (NEO) (16.2 ± 2%) ans saxitoxin (STX) (17.8 ± 2%). The cellular toxicity (mean ± SD: 58.8 ± 7 pg STXeq cell^-1) was essentially independent of light, salinity and nitrate concentration throughout the exponential growth phase, but varied over the growth stages in cultures. A positive correlation was observed between cellular toxicity and salinity-dependent growth rate, indicating that cell toxin quota may be affected by extrinsic factors, but it is not always a direct functional response to specific environmental stress.      

Effects of light intensity variation on nitrogen and phosphorus contents,allocation and limitation in five shade-enduring plants

Aims To enhance the understanding on nitrogen (N) and phosphorus (P) physiological responses to different light environments in shade-enduring plants and provide references to improve the stand structure and ecosystem functions of plantation forests.Methods We selected seedlings of five shade-enduring species with high ecological and economic value in subtropical area of China to study the effects of light intensity on leaf N and P contents, allocation and nutrient limitation in shade-enduring plants. A light intensity gradient of five different levels was set to simulate the varying understory light environment.Important findings With decreasing light intensity, the total biomass and total N and P accumulation of five shade-enduring plants all showed a decreasing trend, but N, P contents in different organs increased. Among them, Gardenia jasminoides (GJ) had the highest while Illicium henryi (IH) had the lowest N content; The P contents of Quercus phillyraeoides (QP) and GJ were significantly higher than Elaeocarpus sylvestris (ES), Ardisia crenata (AC) and IH. QP and GJ had the highest N, P contents under extremely low light intensity (6% natural light intensity) condition (LIC), while AC and IH had the highest N and P contents in low (15% natural light intensity) and moderate (33% and 52% natural light intensity) LIC. ES demanded differently for LIC on N and P, which were 52% and 6% natural light intensity, respectively. N and P allocation of ES, AC and IH followed leaf > root > stem, but for QP and GJ were root > leaf > stem. Decreasing LIC significantly affected N and P allocation. N content variations shown good consistency among different organs under higher LIC (100% natural light intensity) while distinct variability under lower LIC (15% and 6% natural light intensity) in all five species. Phosphorus contents exhibited good consistency in IH, QP and GJ but varied in ES and AC. Decreasing LIC significantly affected organ N/P ratios of shade-enduring plants, but the fundamental growth restriction patterns remained. Light intensity variation and tree species co-regulated N, P utilization and allocation in shade-enduring plants, and then affected the total biomass and total N, P accumulation, which might result from the change of N and P utilization strategy. Therefore, light intensity preference and N, P nutrient balances in shade-enduring plants should be taken into account when constructing multiple layer and uneven-aged forests.     

N、P营养盐对海洋卡盾藻（Chattonella marina）生长的影响

在实验室条件下,设置不同的N、P浓度,研究N、P双因子限制(N:5-500ug·L-1,P:0.74-74ug·L-1,N:P=15)及单因子限制(N:500ug·L-1,P:0.74-741ug·L-1和P:74ug·L-1,N:5-500ug·L-1)对海洋卡盾藻生长的影响.结果表明:在高N、P浓度(N:500ug·L-1,P:74ug·L-1)条件下海洋卡盾藻具有相对较高的比生长率(O.788/d),稳定期持续时间较长,最大细胞密度可达8850 cells/ml.N、P限制能明显抑制海洋卡盾藻的生长,而N限制对海洋卡盾藻生长影响更大,N限制组的比生长率和稳定期细胞密度均明显低于P限制组和N、P双因子限制组.结果说明海洋卡盾藻生长对N变化更为敏感,但随着海洋污染的加剧,海水中N含量持续上升,卡盾藻可迅速爆发性增长并引发赤潮,这也许是近年来我国沿海卡盾藻赤潮频繁发生的重要原因之一.     

Effects of nutrient and light limitation on the biochemical composition of phytoplankton

Three marine phytoplankters (Isochrysis galbana, Chaetoceros calcitrans andThalassiosira pseudonana), commonly used in the culture of bivalve larvae, were grown in batch or semi-continuous cultures. Changes in protein, carbohydrate, lipid and some fatty acids were measured as growth became limited by nitrogen, silicon, phosphorus or light. Under N starvation (2 d) the % lipid remained relatively constant, while% carbohydrate increased and% protein decreased in all 3 species compared to cells growing under no nutrient limitation. Under Si starvation (6 h) there was no change in lipid, protein or carbohydrates. The amount of two fatty acids, 20 : 53 and 22 : 63 remained relatively constant under N, P and Si starvation, exept for a sharp drop in the cells of P-starvedT. pseudonana. However, there were pronounced species differences withI. galbana containing significantly less 20 : 5 3 thanC. calcitrans orT. pseudonana. Under light limitation the amount of lipid per cell showed no consistent trend over a range of irradiances for all 3 species. The amount of N per cell (an index of protein content) as a function of irradiance, was relatively constant forI. galbana andT. pseudonana, while the amount of N per cell was lower under low irradiances forC. calcitrans. These examples of changes in protein, carbohydrate, lipid and certain fatty acids under nutrient (N, Si or P) or light limitation, emphasize the importance of knowing the phase (e.g. logarithmic vs stationary) of the growth curve in batch cultures, since the nutritional value of the phytoplankters could change as cultures become dense and growth is terminated due to nutrient or light limitation.Presented at the XIIIth International Seaweed Symposium, University of British Columbia, Vancouver, Canada, August 1989.     

Effects of enhanced nitrogen deposition and phosphorus limitation on nitrogen budgets of semi-natural grasslands

Increased reactive atmospheric N deposition has been implicated in floristic changes in species‐rich acidic and calcareous grasslands, but the fate of this pollutant N in these ecosystems is unknown. This paper reports the first analysis of N budgets and N fluxes for two grasslands in the White Peak area of Derbyshire, one of the most heavily N‐polluted locations in the UK. N fluxes were monitored in lysimeter cores (retaining the original turfs) taken from field plots of unimproved acidic and calcareous grasslands that had received (in addition to ambient N deposition) simulated enhanced N deposition treatments of 3.5 and 14 g N m^?2 yr^?1 for 6 years. The influence of reducing phosphorus limitation was assessed by factorial additions of P. Seasonal leached losses of nitrate, ammonia and organic N were monitored in detail along with estimates of N removal through simulated grazing and gaseous losses through denitrification and volatilization. The rates of N fluxes by these pathways were used to create N budgets for the grasslands. Both grasslands were found to be accumulating much of the simulated additional N deposition: up to 89% accumulated in the calcareous grassland and up to 38% accumulated in the acidic grassland. The major fluxes of N loss from these grasslands were by simulated grazing and leaching of soluble organic N (constituting 90% of leached N under ambient conditions). Leached inorganic N (mainly nitrate) contributed significantly to the output flux of N under the highest N treatment only. Loss of N through ammonia volatilization accounted for less than 6% of the N added as simulated deposition, while denitrification contributed significantly to output fluxes only in the acidic grassland during winter. The implications of the results for ecosystem N balances and the likely consequences of N accumulation on these grasslands are discussed.     

Green light for the cell cycle

In recent years, considerable progress has been made in unraveling the control mechanisms operating on the plant cell cycle and most of the key regulators have now been identified, including cyclin-dependent kinases (CDKs), cyclins, CDK-inhibitory proteins, the WEE kinase and proteins of the retinoblastoma-related protein (RBR)/E2F/DP pathway. The review discusses recent developments in our understanding of the plant cell cycle machinery and highlights the role of the cell cycle in plant development.