Centric Diatom Thalassiosira Weissflogii Research Articles

Light history modulates growth and photosynthetic responses of a diatom to ocean acidification and UV radiation

To examine the synergetic effects of ocean acidification (OA) and light intensity on the photosynthetic performance of marine diatoms, the marine centric diatom Thalassiosira weissflogii was cultured under ambient low CO2 (LC, 390 μatm) and elevated high CO2 (HC, 1000 μatm) levels under low-light (LL, 60 μmol m−2 s−1) or high-light (HL, 220 μmol m−2 s−1) conditions for over 20 generations. HL stimulated the growth rate by 128 and 99% but decreased cell size by 9 and 7% under LC and HC conditions, respectively. However, HC did not change the growth rate under LL but decreased it by 9% under HL. LL combined with HC decreased both maximum quantum yield (FV/FM) and effective quantum yield (ΦPSII), measured under either low or high actinic light. When exposed to UV radiation (UVR), LL-grown cells were more prone to UVA exposure, with higher UVA and UVR inducing inhibition of ΦPSII compared with HL-grown cells. Light use efficiency (α) and maximum relative electron transport rate (rETRmax) were inhibited more in the HC-grown cells when UVR (UVA and UVB) was present, particularly under LL. Our results indicate that the growth light history influences the cell growth and photosynthetic responses to OA and UVR.

NOVEL PROTEINS COMPRISING THE STRAMENOPILE TRIPARTITE MASTIGONEME IN OCHROMONAS DANICA (CHRYSOPHYCEAE)(1).

Two-dimensional (2-D) protein analysis of the mastigoneme fraction of the chromophyte alga Ochromonas danica E. G. Pringsh. showed the presence of several component proteins of the tubular mastigoneme. Adding to the reported gene Ocm1, three new genes (Ocm2, Ocm3, and Ocm4) belonging to the Ocm gene family were isolated using degenerate primers designed from predicted Ocm1 amino acid sequences. The predicted polypeptides encoded by Ocm2, Ocm3, and Ocm4 were smaller in size than Ocm1. However, they shared four highly conserved, cysteine-rich, epithelial growth factor (EGF)-like motifs, potentially involved in protein-protein interaction. In addition, Ocm2, Ocm3, and Ocm4 showed homology to the SIG protein family in the centric diatom Thalassiosira weissflogii (Grunow) Fryxell et Hasle, which is up-regulated during early stages of sexual reproduction. Immunofluorescence analysis with a polyclonal antibody against the partial amino acid sequences of Ocm2, Ocm3, and Ocm4 showed that Ocm2 and Ocm3 were located in the basal segment region of mastigonemes attached on the surface of the anterior flagellum, and that Ocm4 was located within the tubular shaft portion similar to Ocm1.

RELATIONSHIPS BETWEEN GROWTH RATE, CELL SIZE, AND INDUCTION OF SPERMATOGENESIS IN THE CENTRIC DIATOM THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYTA)1

Patterns of changes in cell size, growth rate, and the inducibility of spermatogenesis were followed in eight sub‐clones of two isolates of the centric diatom Thalassiosira weissflogii (Grunow) Fryxell & Hasle grown at saturating light. One isolate originated from Long Island Sound, New York, USA and the other originated from Jakarta Harbor, Indonesia. As expected from previous studies, oscillations between intervals of cell size reduction and cell size enlargement were observed for each sub‐clone. For both isolates, sperm were easily detected, but cells resembling eggs and auxospores were rarely observed and fertilization was not confirmed, suggesting that the observed cell size increases may have resulted from a combination of asexual cell enlargement and rare auxosporulation. The two isolates differed in their minimum and maximum sizes, and the threshold size for the induction of sperm formation. However, the two sets of isolated sub‐clones displayed comparable relationships between growth rate, sperm inducibility, and cell size relative to the minimum, maximum, and threshold sizes. Growth rate increased as cell size decreased during vegetative divisions until the threshold for sperm inducibility was crossed. Below the size threshold for sperm inducibility, growth rate declined as cell size continued to decrease. Smaller cells were susceptible to failure of normal cytokinesis and valve deposition, resulting in the formation of abnormally long and often multinucleate cells. Culture conditions may select against restoration of cell size via auxosporulation due to the relationship between growth rate and cell size.

INDUCTION OF PHOTORESPIRATION BY LIGHT IN THE CENTRIC DIATOM THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE): MOLECULAR CHARACTERIZATION AND PHYSIOLOGICAL CONSEQUENCES1

Glycine decarboxylase (GDC) is a multi‐subunit enzyme that plays a key role in the photorespiratory pathway. A cDNA (GDCT) with a deduced amino acid sequence sharing up to 64% homology and 50% identity with the T‐protein subunit of GDC from land plants was identified from the centric diatom Thalassiosira weissflogii (Grun.) Fryxell et Hasle. The effect of light on transcript abundance was determined using competitive RT‐PCR. GDCT mRNA per cell decreased during prolonged dark incubations and was higher in cells grown at higher light intensities, independent of growth rate. Cells acclimated to a 12:12‐h light:dark cycle maintained the same amount of GDCT transcripts before and after the dark phase, but rapidly increased transcript levels in the light with a peak “mid‐day.” Rapid increases in GDCT message abundance also occurred in response to an increase in irradiance. Transcript abundance of twca1, the gene for carbonic anhydrase, was also measured and displayed a slower and much less pronounced response to changes in light intensity. An HPLC‐based method was used to measure the concentration of the photorespiratory‐specific compound glycolate in the culturemedia. Only cultures shifted from low subsaturating irradiances to the highest light intensity released significant amounts of glycolate. Our results suggest that quantifying GDCT mRNA in conjunction with measurements of glycolate will allow us to monitor both the initiation of photorespiration and its physiological consequences in the marine environment.

False-positive selection identified by ML-based methods: examples from the Sig1 gene of the diatom Thalassiosira weissflogii and the tax gene of a human T-cell lymphotropic virus.

Sexually induced gene 1 (Sig1) in the centric diatom Thalassiosira weissflogii is considered to encode a gamete recognition protein. Sorhannus (2003) analyzed nucleotide sequences of Sig1 using parsimony analysis and the maximum-likelihood (ML)-based Bayesian method for inferring positive selection at single amino acid sites and reported that positively selected sites were detected by the latter method but not by the former. He then concluded that for this type of study, the ML-based method is more reliable than parsimony analysis. Here we show that his results apparently represent false-positive cases of the ML-based method and that there is no solid evidence that this gene contains positively selected sites. We further demonstrate that in the tax gene of human T-cell lymphotropic virus type I (HTLV-I), all codon sites, including invariable sites, can be inferred as positively selected sites by the ML-based method. These observations indicate that the ML-based method may produce many false-positive sites. One of the main reasons for the occurrence of false positives is that in the ML-based method, codon sites are grouped into several categories, with different nonsynonymous/synonymous rate ratios (omegas), on a purely statistical basis, and positive selection is inferred indirectly by examining whether the average omega for each category is greater than 1. In parsimony analysis, however, the evolutionary change of nucleotides at each codon site is examined. For this reason, parsimony-based methods rarely produce false positives and are safer than ML-based methods for detecting positive selection at individual codon sites, although a large number of sequences are necessary.

IDENTIFICATION OF A GLYCINE DECARBOXYLASE GENE IN THALASSIOSIRA WEISSFLOGII: A PROBE FOR PHOTORESPIRATION IN DIATOMS

Diatoms are responsible for a large percentage of CO2 fixation in the world's oceans. The export of diatom‐fixed carbon from the euphotic zone represents a major sink of CO2 in the global C cycle. Under conditions of high light and high O2/CO2, carbon fixation may be reduced due to photorespiration. The fixation of O2 in photorespiration produces low molecular weight metabolites that cannot be used in the Calvin cycle and are either excreted from the cell or inefficiently salvaged in the photorespiratory pathway. One of the key enzymes of photorespiration is glycine decarboxylase (GDC). We have cloned and sequenced a cDNA corresponding to the gene encoding the T‐protein of GDC from the centric diatom Thalassiosira weissflogii. This cDNA is 1100 bp and its predicted amino acid sequence shows a 45&‘ercnt; identity and a 62% homology with the T‐protein from higher plants. The first 20 amino acids display characteristics of mitochondrial transit peptides, consistent with the localization of GDC in the mitochondria of higher plants. Using competitive RT‐PCR we have demonstrated that transcription of this gene is light‐dependent. Cells maintained at constant growth‐saturating light conditions have much higher levels of T‐protein message than cells kept in the dark for 25 hours. We are currently investigating the effect of other environmental parameters, such as O2 concentration, on T‐protein gene transcription. Ultimately, the goal is to use this gene as a probe for diatom photorespiration in the field. To this end, we are developing degenerate primers and sequencing this gene from other diatom species.

Transformation of Nonselectable Reporter Genes in Marine Diatoms

: We report the genetic transformation of two marine diatoms by microparticle bombardment. The pennate diatom Phaeodactylum tricornutum was transformed with the bacterial gene Sh ble from Streptoalloteichus hindustanus, which confers resistance to the antibiotics phleomycin and zeocin. Transformants contained between 1 and 10 copies of the exogenous DNA integrated into the genome by illegitimate recombination at apparently random locations. Transformation efficiencies were around 10(-6), and individual cell lines could be maintained at -80 degrees C following cryopreservation. Also, P. tricornutum could be transformed simultaneously with two different plasmids, one containing the Sh ble gene and another containing the firefly luciferase gene (LUC) under control of a promoter derived from a fucoxanthin, chlorophyll a/c-binding protein gene (FCP). In these cotransformants, LUC activity was light inducible. The transient transformation of the centric diatom Thalassiosira weissflogii with the bacterial beta-glucuronidase (GUS) gene has also been achieved using similar transformation technology. The availability of gene transfer protocols for marine diatoms, together with a range of functional reporter genes and regulated expression systems, will permit molecular dissection of their biology and allow an assessment of the biotechnological potential of these organisms.

Oxidative transformation of a naturally occurring okadaic acid diol ester by the diatom Thalassiosira weissflogii

The diarrhetic shellfish poisoning (DSP) toxin okadaic acid ( 1) is found in extracts of the dinoflagellate Prorocentrum lima together with a suite of diol esters such as 2. When 2 was added to the culture medium of the centric diatom Thalassiosira weissflogii, it was transformed into a range of products within three days. Three of these products, 3, 4 and 5, were isolated from the medium and identified as oxidation products 2, suggesting that the diatom produces DSP metabolites of greater polarity as a means of detoxification. In addition, another diol ester product 6 was identified as a minor impurity of 2.

Characterization of ferredoxin and flavodoxin as markers of iron limitation in marine phytoplankton

Under conditions of iron stress, many organisms replace the common iron-sulfur redox protein ferredoxin with flavodoxin, a functionally equivalent, non-iron-containing protein. These 2 proteins have been proposed to be indicators of iron nutritional status in marine phytoplankton, but little is known of their expression and regulation. This study characterized their expression by: testing 17 marine phytoplankton isolates from 4 different algal classes for their ability to induce flavodoxin under iron limitation, determining the effect of ecologically relevant limiting factors (other than iron) on flavodoxin expression using the marine centric diatom Thalassiosira weissflogii as a model organism, and examining, in detail, the relationship between iron availability and relative ferredoxin/flavodoxin abundance again using T weissflogii as a model. In the organisms examined, the most common response (1 of 17 isolates ) to iron limitation was induction of flavodoxin and suppression of ferredoxin expression. The remaining 5 isolates, largely of coastal origin, were never observed to produce flavodoxin. These 5 non-inducing organisms have been shown to have high intrinsic Fe requirements for growth and should therefore not present a problem for field measurements of ferredoxin and flavodoxin in iron-poor areas. Expression of flavodoxin in T. weissflogii was found to be specific to iron limitation, and was not induced by nitrate, phosphate, silicate, zinc or light deficiency. The prevalence of the flavodoxin response and its insensitivity to other limiting factors support its use as an indicator of the presence of iron limitation. Iron regulation of relative ferredoxin and flavodoxin abundance (the Fd index) and iron availability was examined in greater detail by measuring the Fd index in T. weissflogii grown over a range of limiting iron concentrations. The relationship between Fd index and growth rate (a proxy for iron availability) is composed of 2 distinct regions. In the first region, at low growth rates, ferredoxin is un<letectable and the Fd index is uniformly zero. In the second region, at moderate-to-fast growth rates, ferredoxin and flavodoxin co-occur in the cells. This implies that flavodoxin substitution is not a simple 'on-or-off' response. Flavodoxin expression is also very sensitive to iron limitation, occurring even at fast growth rates (80 to 90% μ max ). When the 2 proteins co-occurred in cells, their relative abundance (the Fd index) tended to increase along with increasing iron availability. Thus, variation in the Fd index has the potential to indicate spatial and temporal changes in the severity of iron stress in the phytoplankton community.

FLOW CYTOMETRIC ANALYSIS OF SPERMATOGENESIS IN THE DIATOM THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE)1

ABSTRACTFlow cytometry was used to detect and quantify sexual differentiation in the centric diatom Thalassiosira weissflogii (Grun.). Size (light scatter), chlorophyll, protein and DNA contents were measured for each cell throughout the process of differentiation. Male gametes were small round cells characterized by one complement of DNA and a lower protein and chlorophyll content than vegetative cells. Male gamete formation was induced by a long period of darkness (2 days) followed by a transfer to continuous light. Up to 30% of the initial cell population produced male gametes which appeared in the culture 14 h after release from darkness. Male gamete production was also detected in exponentially growing cultures in continuous light, but to a much smaller degree.

FLOW CYTOMETRIC ANALYSIS OF SPERMATOGENESIS IN THE DIATOM THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE)1

ABSTRACTFlow cytometry was used to detect and quantify sexual differentiation in the centric diatom Thalassiosira weissflogii (Grun.). Size (light scatter), chlorophyll, protein and DNA contents were measured for each cell throughout the process of differentiation. Male gametes were small round cells characterized by one complement of DNA and a lower protein and chlorophyll content than vegetative cells. Male gamete formation was induced by a long period of darkness (2 days) followed by a transfer to continuous light. Up to 30% of the initial cell population produced male gametes which appeared in the culture 14 h after release from darkness. Male gamete production was also detected in exponentially growing cultures in continuous light, but to a much smaller degree.

Feeding behaviour in Meganyctiphanes norvegica (M. Sars) (Crustacea: Euphausiacea)

Meganyctiphanes norvegica (M. Sars) will feed upon the centric diatom Thalassiosira weissflogii (Grunow) G. Fryxell & Hasle but cannot fulfil its energy requirement for metabolism on this food. Its daily metabolic requirement can be exceeded when the euphausiid feeds upon the copepods Calanus finmarchicus Gunner or Centropages typicus Krøeyer, but not when feeding upon the smaller copepods Pseudocalanus spp. or Acartia spp. When feeding upon a natural copepod assemblage Meganyctiphanes norvegica requires high concentrations of copepods to achieve its metabolic requirements, suggesting that the euphausiid may exploit vertically patchy concentrations of prey. Short-term predation rates on Pseudocalanus spp. were also used to estimate feeding rates. Feeding in Meganyctiphanes norvegica appears to be adapted to a spatially variable food supply and rapid exploitation of food sources concentrated into patches or layers. The filter area of the feeding basket of M. norvegica is proportionally smaller than the filter area of Euphausia superba Dana, but has the same allometric length exponent. The filter area probably reflects the difference between the primarily carnivorous diet of Meganyctiphanes norvegica and herbivorous diet of Euphausia superba.

Preliminary results from the GATE acoustic echo sounder : MANDICS P. A. and F. F. HALL Jr., 1976. Bull. Am. met. Soc., 57 (9): 1142–1147

Meganyctiphanes norvegica (M. Sars) will feed upon the centric diatom Thalassiosira weissflogii (Grunow) G. Fryxell & Hasle but cannot fulfil its energy requirement for metabolism on this food. Its daily metabolic requirement can be exceeded when the euphausiid feeds upon the copepods Calanus finmarchicus Gunner or Centropages typicus Krøeyer, but not when feeding upon the smaller copepods Pseudocalanus spp. or Acartia spp. When feeding upon a natural copepod assemblage Meganyctiphanes norvegica requires high concentrations of copepods to achieve its metabolic requirements, suggesting that the euphausiid may exploit vertically patchy concentrations of prey. Short-term predation rates on Pseudocalanus spp. were also used to estimate feeding rates. Feeding in Meganyctiphanes norvegica appears to be adapted to a spatially variable food supply and rapid exploitation of food sources concentrated into patches or layers. The filter area of the feeding basket of M. norvegica is proportionally smaller than the filter area of Euphausia superba Dana, but has the same allometric length exponent. The filter area probably reflects the difference between the primarily carnivorous diet of Meganyctiphanes norvegica and herbivorous diet of Euphausia superba.