Uptake of Nitrate by the Diatom Phaeodactylum tricornutum

Ammonium-grown cells of P. tricornutum lack ability to takeup nitrate. This ability develops during 3 h of nitrogen-deprivation;development requires concurrent photosynthesis, and is inhibitedby cycloheximide. Nitrate is accumulated by cells with a developednitrate uptake mechanism. Nitrate uptake is inhibited by 10^–5M CCCP and, in darkness, by anaerobiosis; it therefore presumablyrequires ATP which can be supplied by either photophosphorylationor oxidative phosphorylation.     

Photosynthetic Enhancement in the Diatom Phaeodactylum tricornutum

Enhancement phenomena in photosynthesis of the diatom Phaeodactylum tricornutum Lewin were studied by means of a Haxo oxygen electrode and 2 monochromatic light beams. It was necessary to correct for a minor non-linearity in rate oxygen evolution vs. intensity similar to that reported for Chlorella. Action spectra on complementary backgrounds and derived enhancement spectra were compared to in vivo absorption spectra in identifying the character of pigment systems 1 and 2. Fucoxanthin, chlorophyll c, and chlorophyll a-670 are clearly assignable to pigment system 2 which absorbs in excess at wavelengths 400 to 678 mμ. Chlorophyll a-1 (and a portion of the fucoxanthin) are assignable to pigment system 1 which absorbs in excess at wavelengths 678 to 750 mμ. Enhancement values were generally lower than those observed in Chlorella or Anacystis and lead to conclusion that diatom pigmentation provides an effective light harvesting apparatus.     

The Effects of Excess Irradiance on Photosynthesis in the Marine Diatom Phaeodactylum tricornutum

The response of Phaeodactylum tricornutum to excess light was remarkably similar to that observed in higher plants and green algae and was characterized by complex changes in minimal fluorescence yields of fully dark-adapted samples and declines in maximum variable fluorescence levels and oxygen evolution rates. In our study the parallel decreases in the effective rate constant for photosystem II (PSII) photochemistry, the variable fluorescence yield of a dark-adapted sample, and light-limited O2 evolution rates after short (0-10 min) exposures to photoinhibitory conditions could not be attributed to damage or down-regulation of PSII reaction centers. Instead, these changes were consistent with the presence of nonphotochemical quenching of PSII excitation energy in the antennae. This quenching was analogous to that component of nonphotochemical quenching studied in higher plants that is associated with photoinhibition of photosynthesis and/or processes protecting against photoinhibition in that it did not relax readily in the dark and persisted in the absence of a bulk transthylakoid proton gradient. The quenching was most likely associated with photoprotective processes in the PSII antenna that reduced the extent of photoinhibitory damage, particularly after longer exposures. Our results suggest that a large population of damaged, slowly recovering PSII centers did not form in Phaeodactylum even after 60 min of exposure to excess actinic light.     

Potassium and Photosynthesis in the Marine Diatom Phaeodactylum tricornutum as Related to Washes with Sodium Chloride

Three washes of Phaeodactylum tricornutum with potassium free sodium chloride solution reduced the potassium content of the cells to approximately 7% of the control value at pH 7.0. There was a concomitant reduction of the light induced evolution of oxygen to a value of approximately 20% of the control value. This reduction was less at pH 8.0. Addition of potassium to the washed cells gave rise, after 15 min, to a partial regain of the photosynthetic activity and of cellular potassium. Activities of the two photosystems as assayed here were not dependent on the maintainance of a high potassium content in the cells. The level of ribulose-1,5-diphosphate carboxylase activity was the same whether the cells had been washed with potassium free or potassium containing saline. Ruptured cells rapidly lost their ability to catalyse the photoreduction of dichlorophenolindophenol by water.     

Photochemical and Nonphotochemical Fluorescence Quenching Processes in the Diatom Phaeodactylum tricornutum

Nonphotochemical fluorescence quenching was found to exist in the dark-adapted state in the diatom Phaeodactylum tricornutum. Pretreatment of cells with the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) or with nigericin resulted in increases in dark-adapted minimum and maximum fluorescence yields. This suggests that a pH gradient exists across the thylakoid membrane in the dark, which serves to quench fluorescence levels nonphotochemically. The physiological processes involved in establishing this proton gradient were sensitive to anaerobiosis and antimycin A. Based on these results, it is likely that this energization of the thylakoid membrane is due in part to chlororespiration, which involves oxygen-dependent electron flow through the plastoquinone pool. Chlororespiration has been shown previously to occur in diatoms. In addition, we observed that cells treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea exhibited very strong nonphotochemical quenching when illuminated with actinic light. The rate and extent of this quenching were light-intensity dependent. This quenching was reversed upon addition of CCCP or nigericin and was thus due primarily to the establishment of a pH gradient across the thylakoid membrane. Preincubation of cells with CCCP or nigericin or antimycin A completely abolished this quenching. Cyclic electron transport processes around photosystem I may be involved in establishing this proton gradient across the thylakoid membrane under conditions where linear electron transport is inhibited. At steady state under normal physiological conditions, the qualitative changes in photochemical and nonphotochemical fluorescence quenching at increasing photon flux densities were similar to those in higher plants. However, important quantitative differences existed at limiting and saturating intensities. Dissimilarities in the factors that regulate fluorescence quenching mechanisms in these organisms may account for these differences.     

Effects of Metal Combinations on the Production of Phytochelatins and Glutathione by the Marine Diatom Phaeodactylum tricornutum

Copper, Cd and Zn can be found at elevated concentrations in contaminated estuarine and coastal waters and have potential toxic effects on phytoplankton species. In this study, the effects of these metals on the intracellular production of the polypeptides phytochelatin and glutathione by the marine diatom Phaeodactylum tricornutum were examined in laboratory cultures. Single additions of Cu and Cd (0.4 μM Cu² and 0.45 μM Cd²⁺) to the culture medium induced the production of short-chained phytochelatins ((γ-Glu-Cys)_n-Gly where n = 2–5), whereas a single addition of Zn (2.2 μM Zn²⁺) did not stimulate phytochelatin production. Combination of Zn with Cu resulted in a similar phytochelatin production compared with a single Cu addition. The simultaneous exposure to Zn and Cd led to an antagonistic effect on phytochelatin production, which was probably caused by metal competition for cellular binding sites. Glutathione concentrations were affected only upon exposure to Cd (85% increase) or the combination of Cd with Zn (65% decrease), relative to the control experiment. Ratios of phytochelatins to glutathione indicated a pronounced metal stress in response to exposures to Cu or Cd combined with Zn. This study indicates that variabilities in phytochelatin and glutathione production in the field can be explained in part by metal competition for cellular binding sites.     

Methylcrotonyl-CoA Carboxylase Regulates Triacylglycerol Accumulation in the Model Diatom Phaeodactylum tricornutum

The model marine diatom Phaeodactylum tricornutum can accumulate high levels of triacylglycerols (TAGs) under nitrogen depletion and has attracted increasing attention as a potential system for biofuel production. However, the molecular mechanisms involved in TAG accumulation in diatoms are largely unknown. Here, we employed a label-free quantitative proteomics approach to estimate differences in protein abundance before and after TAG accumulation. We identified a total of 1193 proteins, 258 of which were significantly altered during TAG accumulation. Data analysis revealed major changes in proteins involved in branched-chain amino acid (BCAA) catabolic processes, glycolysis, and lipid metabolic processes. Subsequent quantitative RT-PCR and protein gel blot analysis confirmed that four genes associated with BCAA degradation were significantly upregulated at both the mRNA and protein levels during TAG accumulation. The most significantly upregulated gene, encoding the β-subunit of methylcrotonyl-CoA carboxylase (MCC2), was selected for further functional studies. Inhibition of MCC2 expression by RNA interference disturbed the flux of carbon (mainly in the form of leucine) toward BCAA degradation, resulting in decreased TAG accumulation. MCC2 inhibition also gave rise to incomplete utilization of nitrogen, thus lowering biomass during the stationary growth phase. These findings help elucidate the molecular and metabolic mechanisms leading to increased lipid production in diatoms.     

Highly Efficient Transformation of the Diatom Phaeodactylum tricornutum by Multi-Pulse Electroporation

In our previous studies, a strain of the nonpathogenic, anaerobic, intestinal bacterium, Bifidobacterium longum (B. longum), was found to be localized selectively and to proliferate within solid tumors after systemic administration. In addition, B. longum transformed with the shuttle-plasmid encoding the cytosine deaminase (CD) gene expressed active CD, which deaminated the prodrug 5-fluorocytosine (5-FC) to the anticancer agent 5-fluorouracil (5-FU). We also reported antitumor efficacy with the same plasmid in several animal experiments. In this study, we constructed a novel shuttle-plasmid, pAV001-HU-eCD-M968, which included the mutant CD gene with a mutation at the active site to increase the enzymatic activity.

In addition, the plasmid-transformed B. longum produces mutant CD and strongly increased (by 10-fold) its 5-FC to 5-FU enzymatic activity. The use of B. longum harboring the new shuttle-plasmid increases the effectiveness of our enzyme/prodrug strategy.     

Light-Harvesting Function in the Diatom Phaeodactylum tricornutum: I. Isolation and Characterization of Pigment-Protein Complexes

Three pigment-protein complexes were isolated from the marine diatom Phaeodactylum tricornutum (Bohlin) by treatment of thylakoid membrane fragments with 1% Triton X-100 at 4°C followed by centrifugation on sucrose density gradients. The major complex contains chlorophyll a, c₁, c₂, and the carotenoid fucoxanthin (chlorophyll a: c₁: c₂: fucoxanthin = 1.0: 0.09: 0.28: 2.22) bound to an apoprotein doublet of 16.4 and 16.9 kilodaltons. This complex accounts for >70% of the total pigment and 20 to 40% of the protein in the thylakoid membranes. Efficient coupling of chlorophyll c and fucoxanthin absorption to chlorophyll a fluorescence supports a light-harvesting function for the complex. A minor light-harvesting complex containing chlorophyll a, c₁, and c₂ but no fucoxanthin (chlorophyll a: c₁: c₂ = 1.0: 0.23: 0.26) was also isolated at Triton: chlorophyll a ratios between 20 and 40. These pigments are bound to a similar molecular weight apoprotein doublet. The third complex isolated was the P700-chlorophyll a protein, the reaction center of photosystem I, which showed characteristics similar to those isolated from other plant sources. The yield of the chlorophyll a/c-fucoxanthin complex was shown to respond strongly to changes in light intensity during growth, accounting for most of the changes in cellular pigmentation.     

The Uptake of 63Ni by the Diatom Phaeodactylum tricornutum

Kinetic studies of the uptake of ⁶³Ni by cultures of the diatom Phaeodactylum tricornutum Bohlin revealed that the uptake capacity of the cells depended strongly on their metabolic state. Phosphate-starved cells gave saturation-type uptake curves and had low nickel-binding capacity. This was increased markedly by the addition of phosphate to the cultures. When nickel ions were added before or together with the phosphate, the increase in nickel-binding capacity of phosphate-starved cells failed to appear more or less completely. Uptake of phosphate and formation of polyphosphate by the cells were not affected by the addition of nickel to the growth medium. The phosphate dependent synthesis of the principle involved in nickel binding was induced 15–20 h after the addition of phosphate to starved cells. Whether phosphate was directly or indirectly involved in this process could not be established.     

Biosynthesis of fucoxanthin and diadinoxanthin and function of initial pathway genes in Phaeodactylum tricornutum

The biosynthesis pathway to diadinoxanthin and fucoxanthin was elucidated in Phaeodactylum tricornutum by a combined approach involving metabolite analysis identification of gene function. For the initial steps leading to β-carotene, putative genes were selected from the genomic database and the function of several of them identified by genetic pathway complementation in Escherichia coli. They included genes encoding a phytoene synthase, a phytoene desaturase, a ζ-carotene desaturase, and a lycopene β-cyclase. Intermediates of the pathway beyond β-carotene, present in trace amounts, were separated by TLC and identified as violaxanthin and neoxanthin in the enriched fraction. Neoxanthin is a branching point for the synthesis of both diadinoxanthin and fucoxanthin and the mechanisms for their formation were proposed. A single isomerization of one of the allenic double bounds in neoxanthin yields diadinoxanhin. Two reactions, hydroxylation at C8 in combination with a keto-enol tautomerization and acetylation of the 3'-HO group results in the formation of fucoxanthin.     

The Uptake of Nitrite by the Diatom Phaeodactylum: Interactions between Nitrite and Nitrate

Freshly harvested ammonium-grown cells of the diatom Phaeodactylumtricornutum cannot take up nitrite but they acquire the abilityto do so after a period (about 3 h) of nitrogen deprivationunder conditions that allow photosynthesis. Addition of cycloheximide(1.0 µg ml^–1) prevents development as does incubationin Na⁺ or K⁺-free medium. Nitrite uptake is dependent on thepresence of Na⁺ in the medium and is inhibited by ammonium andnitrite uptake but it is concluded that the inhibition of nitriteuptake by nitrate is not of the competitive type.     

Light-Harvesting Function in the Diatom Phaeodactylum tricornutum: II. Distribution of Excitation Energy between the Photosystems

The distribution of excitation energy between photosystems I and II (PSI and PSII) was investigated in the marine diatom Phaeodactylum tricornutum (Bohlin) using light-induced changes in fluorescence yield and rate of modulated O₂ evolution. The intensity dependence of the fast fluorescence rise in dark adapted cells (±DCMU) suggests that light absorbed by the major antenna complex was not delivered preferentially to PSII but is more equally distributed between the photosystems. Reversible, slow fluorescence yield changes measured in the absence of DCMU were correlated with decreased initial fluorescence and rate constants for PSII photochemistry, increased variable fluorescence, alteration of the fluorescence excitation and emission spectra, and could be effected by either 510 nm (PSII) or 704 nm (PSI) light. Slow, reversible fluorescence yield changes were also observed in the presence of DCMU, but were characterized by a loss of both initial and variable fluorescence and could not be induced by PSI light. The absence of slow changes in the yield of fluorescence and rate of modulated O₂ evolution, following addition or removal of PSI background light to modulated PSII excitation, does not support regulation of excitation energy density in PSI at the expense of PSII. The results suggest that adjustments are made at the level of excitation energy transfer to the PSII reaction center which prevent prolonged loss of photosynthetic capacity. Energy distribution is regulated by ionic distributions independently of the plastoquinone pool redox state. These differences in light-harvesting function are probably a response to the aquatic light field and may account for the success of diatoms in low and variable light environments.     

Activation of Diadinoxanthin De-Epoxidase Due to a Chiororespiratory Proton Gradient in the Dark in the Diatom Phaeodactylum tricornutum

Abstract: An exponential dynamic light regime with prolonged dark periods (light/dark cycle 8/40 h) was used to simulate deep mixing of algae in natural waters and to investigate the adaptation of the diatom Phaeodactylum tricornutum to these extreme light conditions. After prolonged dark periods Phaeo dactylum cells showed surprisingly high contents of diatoxan-thin, low photosynthetic efficiency and high non-photochemical quenching (NPQ) of chlorophyll fluorescence. Diatoxanthin con centrations and NPQ were low at the beginning of the dark peri od and increased with the duration of the dark incubation. Addi tion of the diadinoxanthin de-epoxidase inhibitor, DTT, prevent ed the formation of diatoxanthin, thereby excluding de novo synthesis of diatoxanthin during the prolonged dark period. Evi dence of chlororespiratory electron flow and the establishment of a diadinoxanthin de-epoxidase activating proton gradient in the dark was derived from two observations. First, uncoupling of electron transport with NH₄CI at the beginning of the dark period prevented the development of non-photochemical quenching of chlorophyll fluorescence and the formation of diatoxanthin during the dark period. Second, inhibition of the electron and proton consuming terminal redox component of chlororespiratory electron transport, cytochrome oxidase, by addition of KCN induced stronger NPQ and a higher de-epoxidation state of the xanthophyll cycle. These results strongly indi cate that the activation of diadinoxanthin de-epoxidase in the dark is the consequence of a chlororespiratory proton gradient. We furthermore present evidence that diatoxanthin formed by the chlororespiratory proton gradient has the same efficiency in the mechanism of enhanced heat dissipation as diatoxanthin induced by a light-driven ApH.     

Polypeptides of a Light-Harvesting Complex of the Diatom Phaeodactylum tricornutum Are Synthesized in the Cytoplasm of the Cell as Precursors

A light-harvesting fucoxanthin-chlorophyll a/c-protein complex has been isolated from the diatom Phaeodactylum tricornutum by detergent extraction of thylakoid membranes coupled with sucrose density gradient centrifugation. The isolated complex was devoid of photochemical activity and displayed spectral characteristics consistent with light harvesting function. It has three major polypeptides of apparent molecular weights 18,000, 19,000, and 19,500 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Using protein synthesis inhibitors, these polypeptides were shown to be synthesized on 80S cytoplasmic ribosomes. Antibodies raised to a mixture of the 19,000 and 19,500 dalton components of the complex were used to demonstrate structural similarity among the three polypeptide components. Immunoprecipitation from primary translation products synthesized in a reticulocyte lysate system primed with P. tricornutum poly(A) RNA, indicates that the polypeptide components are synthesized as precursors 3,000 to 5,000 daltons larger than the mature polypeptides.