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Applied and Environmental Microbiology, June 2000, p. 2349-2357, Vol. 66, No. 6
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Molecular and Physiological Responses of Two
Classes of Marine Chromophytic Phytoplankton (Diatoms and
Prymnesiophytes) during the Development of Nutrient-Stimulated
Blooms
Michael
Wyman,1,*
John T.
Davies,1
David W.
Crawford,2,
and
Duncan A.
Purdie3
Department of Biological Sciences, University
of Stirling, Stirling FK9 4LA,1 and
School of Ocean and Earth Sciences, University of Southampton,
Southampton Oceanography Centre, Southampton SO14
3ZH,3 United Kingdom, and Department of
Earth and Ocean Sciences (Oceanography), University of British
Columbia, Vancouver, Canada V62 1242
Received 26 October 1999/Accepted 13 March 2000
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ABSTRACT |
Generic taxon-specific DNA probes that target an internal region of
the gene (rbcL) encoding the large subunit of
ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) were
developed for two groups of marine phytoplankton (diatoms and
prymnesiophytes). The specificity and utility of the probes were
evaluated in the laboratory and also during a 1-month mesocosm
experiment in which we investigated the temporal variability in RubisCO
gene expression and primary production in response to inorganic
nutrient enrichment. We found that the onset of successive bloom events
dominated by each of the two classes of chromophyte algae was
associated with marked taxon-specific increases in rbcL
transcription rates. These observations suggest that measurements of
RubisCO gene expression can provide an early indicator of the
development of phytoplankton blooms and may also be useful in
predicting which taxa are likely to dominate a bloom.
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INTRODUCTION |
The majority of marine eukaryotic
phytoplankton belong to several rather distantly related classes of
chlorophyll a- and c-containing microalgae known
as chromophytes. These organisms include the diatoms and
prymnesiophytes, and like cyanobacteria and higher plants, the
principal route of photosynthetic CO2 fixation is via the
Calvin cycle enzyme ribulose-1,5-bisphosphate
carboxylase/oxygenase (RubisCO). With the exception of
peridinin-containing dinoflagellates (16, 21, 26), all known
chromophytes produce a form ID RubisCO enzyme encoded by the
chloroplast genes rbcL and rbcS (15, 18, 23). By contrast, the RubisCO produced by many oceanic
picocyanobacteria (Synechococcus and
Prochlorococcus spp.) is related to the form IA enzyme
present in some autotrophic members of the 
and 
subclasses of
Proteobacteria (22, 25, 28), whereas other
cyanobacteria (including the marine diazotroph Trichodesmium
thiebautii) and all chlorophytes (higher plants and green algae)
produce a form IB enzyme.
The restricted phylogenetic distribution of the form IA and IB enzymes
in marine phytoplankton has been exploited recently to examine the
temporal (diel) pattern of RubisCO gene expression in
picoplanktonic cyanobacteria (18, 20, 28). Several
studies have also examined variability in form ID RubisCO gene
expression in natural populations of eukaryotic microphytoplankton
(18, 19, 29, 31). The RubisCO gene probes employed in the
latter studies, however, were not designed to discriminate among the various classes of chromophyte algae. As a result, current
understanding of the temporal and spatial patterns of RubisCO gene
expression in natural populations of marine phytoplankton is less
developed for chromophytes than for the picocyanobacteria.
In contrast to the picocyanobacteria, microplanktonic chromophytes such
as chain-forming diatoms play a major role in the drawdown of
atmospheric CO2 (the so-called biological pump)
(14). Calcifying prymnesiophytes (such as Emiliania
huxleyi) are responsible for much of the biologically mediated
inorganic carbon flux to the deep ocean (4). Therefore, it
is of considerable practical importance to understand the environmental
factors which regulate RubisCO synthesis (and hence photosynthetic
carbon fixation and productivity) in these widely distributed and
biogeochemically important groups of marine phytoplankton. As a first
step toward this end, we developed taxon-specific RubisCO gene probes
for the diatom and prymnesiophyte classes of marine chromophytes. Here
we present the results of field trials conducted during the PRIME
(Plankton Reactivity in the Marine Environment) mesocosm study
(27). Our main objective during this 1-month experiment was
to investigate the effects of inorganic nutrient enrichment on the
temporal dynamics of diatom and prymnesiophyte RubisCO gene expression,
photosynthetic carbon fixation, and the growth and development of
phytoplankton blooms dominated by members of these two classes of
marine chromophytes.
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MATERIALS AND METHODS |
Mesocosms.
Eight transparent polyethylene enclosures
(diameter, 2 m; depth, 4.25 m) were attached to the southern
side of a floating raft in an embayment of the Raunefjorden 200 m
offshore at the University of Bergen Espegrend field station. On 6 June
1995 each enclosure was filled with 11 m3 of unfiltered
near-surface (depth, 1 m) seawater pumped from below the raft. The
following day, several of the mesocosms were supplemented with
additional inorganic nutrients; one enclosure was supplemented with 15 µM nitrate, 1 µM phosphate, and 39 µM silicate
(N/P/Si-supplemented enclosure), and another was amended with 15 µM
nitrate and 1 µM phosphate (N/P-supplemented enclosure). For the
remainder of the experiment (7 June 1995 to 4 July 1995) the water
columns in the enclosures were mixed continuously by using an air
uplift system to maintain a homogeneous vertical distribution of phytoplankton.
Starting at 0730 h each day, 10% (by volume) of the seawater was
removed from the enclosures in order to obtain material for experimental analysis and observation. The mesocosms were replenished with an equal volume (1.1 m3) of fresh seawater amended
with 15 µM nitrate and 1 µM phosphate once sampling had been
completed. The concentrations of nutrients (N, P, Si) in the enclosures
and the surrounding seawater (depth, 1 m) were determined daily by
using a Skalar autoanalyzer and standard analytical procedures
(17). Surface incident irradiance was measured continuously
throughout the experiment with a cosine-corrected PAR sensor and a
Li-Corr model Li-1000 data logger. Additional details concerning the
experimental design, management, and behavior of these enclosures and
six other nutrient-amended mesocosms have been described by Williams
and Egge (27).
Determination of phytoplankton abundance, biomass, and
depth-integrated primary production.
Phytoplankton were identified
and enumerated by using samples collected at a depth of 1 m every
other day and preserved with 0.4% (vol/vol) neutralized formalin and
acid lugol (27). Phytoplankton cell counts were converted to
biomass values (micrograms of C per liter) by using the procedures
described by Eppley et al. (8).
Photosynthesis-irradiance response curves for the phytoplankton
communities in each mesocosm were determined every second
day by the
[
14C]bicarbonate uptake technique by using the
experimental and data
management procedures described by Wyman et al.
(
29). The light
attenuation coefficient was derived by log
linear regression of
in situ measurements of downwelling irradiance
determined at 0.5-m
depth intervals with a submersible cosine-corrected
PAR sensor
and a calibrated Crump or Macam quantum
meter.
Depth-integrated primary production (millimoles of C per square meter
per day) was estimated by using the model of Talling
(
24) by
summation of the calculated rates of carbon fixation
for each 15-min
period throughout the day. These rates were derived
from the
photosynthetic parameters P
max and
given by Wyman et
al. (
29), the prevailing light attenuation coefficient for
each
mesocosm, and concurrent average values for mean surface incident
irradiance for each 15-min
interval.
DNA isolation, PCR amplification, and cloning of rbcL
gene fragments.
DNA was isolated from field-collected samples of
T. thiebautii as previously described (13) and
from laboratory cultures of Thalassiosira pseudonana,
Skeletonema costatum, E. huxleyi, and
Synechococcus sp. strain PCC6301 by using a modified
cetyltrimethylammonium bromide extraction method (1).
Coccolithus pelagicus cells were collected from a natural
population growing in the northeast Atlantic Ocean (cruise D 221, RRS
Discovery, June and July 1996) by aspirating these rapidly sedimenting
phytoplankton cells from an on-deck incubator fed with surface
seawater. Following centrifugation (1,000 × g for
15 s) the pelleted cells were resuspended in 100 mM Tris-HCl (pH
8.0)-100 mM EDTA-250 mM NaCl and stored frozen at 
70°C until
cetyltrimethylammonium bromide extraction and DNA isolation ashore.
Genomic DNA from Prochlorococcus marinus was kindly provided
by D. Scanlan, University of Warwick.
An internal region of
rbcL was amplified from all DNA
samples by using fully degenerate versions of the oligonucleotide
primers
described by Xu and Tabita (
31). The primer pair
used (5'-GCGAATTCAARCCNAARYTNGGNYTNTC-3'
and
5'-AGGGATCCYTCNARYTTNCCNACNAC-3') targets two highly
conserved
motifs (KPKLGLS and VVGKLEG) within the
rbcL genes
of a diverse
range of photoautotrophs (
31). Recognition
sites for restriction
endonucleases
EcoRI and
BamHI are present toward the 5' ends of
the upstream and
downstream primers, respectively. PCR amplification
of
rbcL
was carried out with a Techne Omnigene thermocycler by
using Amplicycle
reagents (Perkin-Elmer Ltd.) in the presence
of 10 to 100 ng of
template DNA, 25 pmol of each primer, and 2
mM MgCl
2. The
PCR cycling parameters were as follows: five cycles
consisting of
95°C for 1 min, 37°C for 1 min, and 72°C for 2 min,
followed by
25 cycles in which a higher annealing temperature
(45 rather 37°C)
was used under otherwise identical reaction
conditions.
PCR products of the expected size (~480 bp) were isolated from
low-melting-point agarose gels and were purified by using a
commercial
kit as recommended by the supplier (Hybaid Ltd.). The
purified
fragments were ligated into the T-tailed plasmid vector
pCR2.1
(Invitrogen Corp.) and were cloned in
Escherichia coli host
strain Inv-
F' supplied with the vector. Plasmid DNA was
isolated
from recombinant colonies, and the identities of the
cloned
rbcL fragments were confirmed by performing nucleotide
sequencing of both strands and comparing the sequences (by using
the
National Center for Biotechnology Information BlastX search
routine)
with known peptide sequences in the GenBank database.
When the
degenerate primer regions were excluded, the nucleotide
sequences of
the
rbcL fragments were identical (
Synechococcus sp. strain PCC 6301,
S. costatum, and
E. huxleyi)
or nearly identical
(
P. marinus) to the sequences determined
previously and deposited
in the GenBank
database.
DNA sequence analysis and development of taxon-specific
rbcL gene probes.
Marine diatom and prymnesiophyte
rbcL nucleotide sequences (including sequences determined in
the present study) were retrieved from the GenBank database, and the
optimal alignment and pairwise levels of identity for the ~480-bp
gene internal region were determined by using Clustal X
(11). Probes were synthesized from the primary rbcL clones isolated from S. costatum (diatom)
and E. huxleyi (prymnesiophyte) by PCR incorporation of
alkali-labile digoxigenin-dUTP (Boehringer Mannheim) by using
oligonucleotide primers targeted to pCR2.1 vector sequences flanking
the cloned inserts. The PCR cycling parameters employed were as
follows: 30 cycles consisting of 95°C for 1 min, 68°C for 1 min,
and 72°C for 1 min, followed by a final extension step consisting of
20 min at 72°C. The taxonomic specificity of each probe was assessed
by Northern analysis of in vitro transcription products synthesized
from the cloned rbcL genes as described below.
The inserts of all seven
rbcL clones produced in this study
were excised from pCR2.1 by restriction endonuclease digestion
with
BamHI and
EcoRI and subcloned in pGEM3Z (Promega
Inc.). Sense
strand transcripts were synthesized in vitro from the T7
promoter
of the vector by using T7 RNA polymerase and the reaction
conditions
recommended by the supplier (Boehringer Mannheim). Following
treatment
of the reaction products with DNase I (RNase-free; Boehringer
Mannheim), the integrity of the transcripts was verified by agarose
gel
electrophoresis, and the yield of each reaction was determined
by UV
spectrophotometry (
1).
Equal quantities of transcription products (50 and 10 ng) were
immobilized on positively charged nylon membranes (Boehringer
Mannheim)
by Northern slot blotting as previously described (
29).
Total RNA (1 and 0.2 µg) from the enteric bacterium
E. coli was
also included in two separate slots on each blot as a
negative
control. After exhaustive preliminary optimization of
hybridization
and posthybridization conditions, blotted membranes were
hybridized
overnight at 42°C in DIG Easy Hyb solution (Boehringer
Mannheim)
amended with 50 ng of denatured probe DNA per ml. Stringency
washes
were performed the following day by rinsing the membranes in 2×
SSPE (1× SSPE is 150 µM NaCl plus 10 µM
Na
2HPO
4 plus 1 µM EDTA)
containing 0.1%
(wt/vol) sodium dodecyl sulfate (SDS) at ambient
temperature and then
washing them twice (30 min each) in 0.5×
SSPE-0.1% (wt/vol) SDS at
50°C.
Hybrids were detected immunochemically with alkaline
phosphatase-conjugated anti-digoxigenin in conjunction with the
chemiluminescent
substrate CDP-Star as recommended by the supplier
(Boehringer
Mannheim). Luminographs were obtained by exposing the
membranes
to Kodak Biomax MR film, and densitometric data were
collected
by using a flat-bed scanner (Hewlett-Packard model 5P) and
the
GelWorks v.2.01 (UVP Ltd.) analysis
package.
RNA isolation and Northern analyses.
Seawater (5 to 14 liters) was obtained from the two nutrient-supplemented enclosures
~4.5 h after sunrise on each day of the experiment, and phytoplankton
cells were collected by gentle filtration onto 90-mm-diameter Whatman
GF/C filters. The filters were placed in 5 ml of ice-cold RNA
extraction buffer (100 mM LiCl, 50 mM Tris-HCl [pH 7.5], 1 mM EGTA,
1% [wt/vol] SDS), snap frozen, and stored at 20°C. At the end of
the experiment the frozen samples were transported to the United
Kingdom on dry ice and extracted in hot acid-phenol as described
previously (30). The purified nucleic acids were taken up in
0.5 ml of DNase buffer (100 mM sodium acetate, 10 mM
MgCl2), and the DNA was hydrolyzed by treatment with 50 U
of DNase (RNase-free; Roche) at 37°C for 1 h. The DNase was
inactivated by phenol-chloroform extraction, and the RNA was pelleted
by ethanol precipitation and taken up in 100 µl of
diethylpyrocarbonate-treated deionized water (30). Following
purification, the integrity of RNA samples was verified by
electrophoresis through formaldehyde agarose gels stained with ethidium
bromide (1). Aliquots (5 µg) of total RNA were prepared
for Northern analysis by using the rbcL probes, blotting
procedures, and optimized hybridization and posthybridization
conditions described above.
Following detection of
rbcL hybrids, each membrane was
washed briefly in 2× SSPE and stripped of digoxigenin by mild alkali
treatment (0.2 M NaOH-0.1% SDS at 37°C for 15 min). The relative
amount of phytoplankton RNA immobilized in each slot was determined
as
previously described (
29) by rehybridizing the membranes
with a 5'-digoxigenin end-labelled oligonucleotide probe
(5'-CTCCCCTAGCTTTCGTCC-3')
targeted to a conserved region in
the chloroplast-encoded 16S
rRNA gene of oxygenic photoautotrophs. This
procedure was adopted
in order to correct for any variability in the
relative amounts
of nonphytoplankton RNA (e.g., RNA derived from
zooplankton) present
in the samples. In practice, however, the
contribution of nonphytoplankton
RNA to the total RNA was not that
variable during the experiment,
and normalization by this procedure was
not required for the majority
of
samples.
Adjustments were also made in order to normalize for the relative
contribution of each taxonomic group (diatoms or prymnesiophytes)
to
the total phytoplankton biomass in the enclosures at the time
of
sampling. For example, if at two different times diatoms comprised
20 and 80% of the biomass, the
rbcL hybridization signals were
normalized by factors of 5- and 1.2-fold, respectively. Following
normalization, the hybridization signals were expressed as percentages
of the maximum signal recorded for each probe type in each of
the
enclosures.
Nucleotide sequence accession numbers.
The three novel
rbcL nucleotide sequences determined in this study have been
deposited in the GenBank database under the following accession
numbers: T. thiebautii, AF136182; C. pelagicus,
AF196307; and T. pseudonana, AF109210.
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RESULTS |
DNA sequence analysis and development of taxon-specific
rbcL gene probes.
At the start of this study we
conducted a preliminary comparison of the few marine diatom and
prymnesiophyte rbcL nucleotide sequences that were then
deposited in the GenBank database. The peptide sequences of the gene
internal regions examined were found to be well conserved, but
alignment of the DNA sequences revealed somewhat greater variability,
particularly between members of the two taxonomic groups. Several more
diatom and prymnesiophyte rbcL gene sequences have been
added to the GenBank database in the intervening years, and
representatives of these sequences were retrieved and included in an
updated alignment (Fig. 1). In agreement
with our earlier findings, a pairwise comparison of these sequences
revealed that the nucleotide identity between the rbcL genes
from members of the two classes ranged from 74 to 79%, whereas
identities of 87 to 90 and 86 to 93% were observed within the diatom
and prymnesiophyte groups, respectively.
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FIG. 1.
Nucleotide sequence alignment of rbcL gene
fragments from marine diatom and prymnesiophyte phytoplankton.
Nucleotides identical to the first sequence in the alignment are
indicated by dashes. The diatom sequences are shaded. The GenBank
accession numbers are as follows: Umbilicosphaera sibogae
D45843; Calyptrosphaera sphaeodea, D45842;
Chrysochromulina hirta, D45846; Emiliania
huxleyi, D45845; Pleurochrysis carterae, D11140;
Coccolithus pelagicus, AF196307; Cylindrotheca
sp. strain N1, M59080; Thalassiosira pseudonana, AF109210;
Skeletonema costatum, AF015569; and Rhizosolenia
setigera, AF015568.
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The conserved nature of the
rbcL coding sequences within
each phytoplankton class prompted us to develop generic
rbcL
probes
for each taxonomic group. The cloned
rbcL genes of
S. costatum (diatom group) and
E. huxleyi
(prymnesiophyte group) were selected
as the sources of the probes, and
we assessed their general suitability
by performing quantitative
Northern blotting of in vitro transcription
products derived from the
rbcL genes of members of both groups.
Following
optimization, each probe produced a similar signal for
a given amount
of target RNA that was both taxon specific and
of equivalent
sensitivity for homologous and near-homologous targets
derived from
members of the same phytoplankton class (Fig.
2).
Equally important, neither probe
hybridized with
rbcL transcription
products from members of
other phytoplankton groups or with the
negative control (total RNA from
E. coli).
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FIG. 2.
Northern slot blots of in vitro transcription products
derived from various species of marine phytoplankton probed with either
the diatom-specific or prymnesiophyte-specific rbcL gene
probes. Each slot was loaded with either 50 or 10 ng of target sense
strand rbcL RNA, whereas the last row of both blots
contained 1 or 0.1 µg of total RNA from the enteric bacterium
E. coli as a nonspecific negative control. The hybridization
and posthybridization conditions employed are described in the text.
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Temporal variability in nutrient concentrations, phytoplankton
biomass, and primary productivity in nutrient-enriched mesocosms.
Nitrate and phosphate concentrations declined rapidly in both
nutrient-enriched enclosures during the first 5 days of the experiment
and thereafter were invariably less than 1.0 and 0.1 µM, respectively
(Fig. 3). Although the initial rate of
silicate utilization in the N/P/Si-supplemented enclosure was somewhat lower than that of either nitrate or phosphate, silicate concentrations decreased much more rapidly after the first 48 h and remained significantly below 0.5 µM after day 6.
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FIG. 3.
(a and b) Temporal variation in the inorganic nutrient
concentrations in the N/P/Si-supplemented enclosure (a) and the
N/P-supplemented enclosure (b) during the PRIME mesocosm experiment.
Symbols:  , silicate;  , nitrate;  , phosphate. The insets show
the dissolved nutrient concentrations (micromolar) in the enclosures
from day 5 onward on an expanded scale (phosphate, ×10). (c) Molar
ratio of N assimilation to P assimilation in the two mesocosms. The
horizontal dotted line indicates the ratio (15:1) of N and P supplied
to the mesocosms.
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Both mesocosms were supplemented and subsequently resupplied with N and
P at near Redfield ratio (15:1), but significant differences
were
apparent in the relative rates at which these nutrients were
assimilated during the course of the experiment (Fig.
3). In the
first
24 h, phosphate uptake was particularly rapid, resulting
in an N/P
assimilation ratio of ~2.5:1 in both enclosures. The
relative rate of
nitrate assimilation increased over the next
few days, however, and by
day 4 (N/P/Si-supplemented mesocosm)
or day 6 (N/P-supplemented
mesocosm) was at almost twice Redfield
ratio. Although there was some
day-to-day variability, the mean
ratio of N assimilation to P
assimilation from the second week
on was close to 15:1 in both
mesocosms (excluding the anomalous
value recorded on day 13 in the
N/P-supplemented enclosure), i.e.,
similar to the proportions of the
two elements supplied (Fig.
3).
Throughout the experiment, the concentration of silicate in the
N/P-supplemented enclosure did not decrease markedly below
the initial
concentration present in the seawater introduced into
the mesocosms at
zero time (Fig.
3). The concentration of silicate
in the surrounding
seawater used to replenish the enclosures gradually
increased after day
10, but at most this would have added an extra
0.12 µM per day to the
mesocosms (data not shown). The ratio of
N uptake to Si uptake in the
silicate-amended mesocosm was approximately
1 for the first 3 days, but
it declined substantially in the next
2 days to 0.23 (day 4) and 0.13 (day 5) as dissolved silicate
was rapidly removed from the enclosure
(Fig.
3). By day 6, however,
the silicate concentration had been
reduced to 0.69 µM, and in
the absence of additional supplements, no
significant Si uptake
occurred after the first
week.
Phytoplankton biomass increased dramatically in both enclosures
following the initial addition of nutrients (Fig.
4 and
5).
In the first enclosure (the N/P/Si-supplemented enclosure) a mixed
bloom dominated by three diatom species (
Leptocyclindricus
danicus,
Pseudonitzschia sp. and
S. costatum) and the prymnesiophyte
E. huxleyi developed.
At the height of the bloom (day 6) silicate
was all but exhausted (Fig.
3), and at this point diatoms accounted
for ~70% of the total
phytoplankton biomass (Fig.
4). The importance
of all three diatom
species gradually declined thereafter until
about day 20, and then,
largely as a result of a net increase
in the
L. danicus
population, a small but significant (approximately
two- to threefold)
increase in biomass occurred over the next
5 days. Prior to
reinitiation of diatom growth, however, a pronounced
and sustained
secondary
E. huxleyi bloom was observed following
the demise
of the mixed primary bloom, and by day 22 this species
accounted for
~80% of the total biomass.
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FIG. 4.
Temporal variation in the abundance of diatom
rbcL mRNA (a), the abundance of prymnesiophyte
rbcL mRNA (b), depth-integrated (0 to 4.5 m)
primary production (c), and phytoplankton biomass
(d) (, diatoms;  , E. huxleyi) in the
N/P/Si-supplemented mesocosm. d, day.
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FIG. 5.
Temporal variation in the abundance of diatom
rbcL mRNA (a), the abundance of prymnesiophyte
rbcL mRNA (b), (c) depth-integrated (0 to 4.5 m) primary production (c), and phytoplankton
biomass (d) (, diatoms; , E. huxleyi) in
the N/P-supplemented mesocosm. d, day.
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As we had anticipated, the initial behavior of the phytoplankton
population in the second enclosure (N/P-supplemented enclosure)
was
distinct from that observed in the first enclosure (Fig.
5).
Although
the diatom biomass doubled during the first 48 h, this
growth
response was not sustained in the absence of added silicate,
and both
the primary and secondary blooms in this enclosure were
dominated by
E. huxleyi. The peak of the primary bloom occurred
somewhat
later (day 8), whereas the reinitiation of net growth
leading to the
secondary
E. huxleyi bloom occurred a little earlier
(~2
to 3 days) than in the first enclosure. The
E. huxleyi
biomass
at the peak of both blooms in the second enclosure was very
similar
to the
E. huxleyi biomass observed during the
secondary bloom
of this prymnesiophyte in the N/P/Si-amended
mesocosm.
After the first day of the experiment, primary production rates
increased to a peak on day 3 and day 5 in the first
(N/P/Si-supplemented)
and second (N/P-supplemented) enclosures,
respectively (i.e.,
~3 days before the biomass maxima were reached in
either mesocosm)
(Fig.
4 and
5). At their maxima, production rates were
as high
as 348.6 mmol of C m
2 day
1 in the
first enclosure and 218.9 mmol of C m
2 day
1
in the N/P-supplemented mesocosm. However, production rates declined
steadily as the blooms peaked and then collapsed in both enclosures.
After day 13 a gradual recovery in primary production rates first
preceded and then presumably sustained the development of the
secondary
blooms. While the rate of primary production was somewhat
higher
(~1.6-fold) at the peak of the primary bloom in the diatom-dominated
enclosure than in the N/P-amended enclosure, very similar but
substantially lower C assimilation rates were recorded during
the
secondary
E. huxleyi-dominated blooms in both
mesocosms.
Temporal variability in diatom and prymnesiophyte rbcL
gene expression.
The initial nutrient supplements stimulated
dramatic increases (~2 orders of magnitude) in the abundance of
diatom and prymnesiophyte rbcL mRNAs in both enclosures
(Fig. 4 and 5). Maximal rbcL expression occurred on day 2 (i.e., 1 or 2 days before the peaks in production and 4 to 6 days
before the height of the primary blooms in the first and second
enclosures, respectively). After day 2, however, the decline in the
abundance of rbcL transcripts produced by members of each
group was equally rapid, and by day 5 the diatom and prymnesiophyte rbcL mRNA levels in both enclosures were similar to those
observed during the first 24 h.
For the remainder of the experiment (day 5 onward), very little change
occurred in the overall abundance of diatom
rbcL mRNA
except
for a transient (but comparatively minor) increase in transcript
levels
after day 19 in the first (N/P/Si-supplemented) enclosure
prior to
regrowth of
L. danicus. In contrast, pronounced increases
in
the abundance of prymnesiophyte
rbcL mRNA were observed in
both mesocosms prior to the development of the secondary blooms
dominated by
E. huxleyi. In the N/P/Si-amended enclosure, a
significant
and sustained increase in prymnesiophyte
rbcL
expression occurred
after day 13, and transcript levels rose steadily
over the following
week to a peak on day 21. The increase in the
abundance of
rbcL mRNA occurred approximately 2 days earlier
in the second (N/P-supplemented)
enclosure, however, and transcript
levels increased more sharply
to reach a double peak on days 13 and 15. Although the timing
of events was somewhat different, similar temporal
sequences were
observed during the development of the secondary blooms
in the
two enclosures. Like the primary blooms, the phytoplankton
biomass
peak occurred some time after the initial increase in the
abundance
of
rbcL mRNA and was preceded by a significant
(albeit less dramatic)
net increase in the rate of primary
production.
| |
DISCUSSION |
Development of taxon-specific rbcL probes for marine
diatom and prymnesiophyte algae.
Previous studies have exploited
the divergent nature of form I RubisCO large-subunit genes to develop
clade-specific rbcL gene probes for the
cyanobacterium-chlorophyte and chromophyte phytoplankton lineages
(18, 19, 31). However, since the major classes of eukaryotic
marine phytoplankton all belong to the chromophyte clade, it has not
been possible until now to obtain taxon-specific information concerning
RubisCO gene expression for phytoplankton other than the oceanic
picoplanktonic cyanobacteria. In this study we developed and
successfully deployed specific rbcL gene probes for two of
the major classes (diatoms and prymnesiophytes) of chromophytic algae.
Apart from nonidentities attributable to conserved and nonconserved
amino acid substitutions, many of the differences between
the
rbcL genes of members of the two chromophyte classes were
found in the P
3 positions of otherwise synonymous codons.
This
suggests that there is a distinct preference in codon usage in
these algae, which, at least in the case of highly expressed genes
like
rbcL, is conserved among members of the same taxonomic
class.
Similar degrees of nonidentity between the
rbcL genes
of the diatom
Cylindrotheca sp. strain N1 and various
rhodophytes and between
the
rbcL genes of haptophytes and
members of several other classes
of marine phytoplankton have been
reported previously (
5,
15).
Differences in
rbcL codon usage have also been found in more
closely related marine phytoplankton. The peptide sequences of
the
novel
rbcL genes present in the marine picoplanktonic
cyanobacteria
Synechococcus sp. strain WH7803 and
P. marinus are highly conserved,
but the corresponding nucleotide
sequences are only 71% identical
(
25). The degree of
third-base degeneracy between the two sequences
is such that the
longest run of identical bases is only 14 bp
long, which is short
enough that a
Synechococcus sp. strain WH7803
rbcL gene probe failed to recognize the
P. marinus homologue in
Southern blots of genomic DNA even under
low-stringency
conditions.
Alignment of the diatom and prymnesiophyte
rbcL nucleotide
sequences revealed that conserved runs consisting of >14 identical
nucleotides were rare except among sequences derived from members
of
the same group. This degree of sequence degeneracy indicated
that it
should be possible to obtain taxon-specific information
concerning
the relative abundance of the
rbcL mRNAs produced by
members
of each class of chromophytes by using generic gene probes
generated
from a single representative of either group. We selected
the
S. costatum and
E. huxleyi rbcL clones as sources of the
diatom
and prymnesiophyte probes, respectively, but in many respects
these choices were arbitrary. The longest run of identical bases
between the two probes is 19 bp long, whereas identical regions
more
than twice this length occur in the diatom probe and the
rbcL gene of
Cylindrotheca sp. strain N1 (41 bp)
and in the prymnesiophyte
probe and
Pleurochrysis carterae
(50 bp), the least similar target
sequences to the probes found in
either
group.
We were able to establish that the probes did not cross-hybridize with
rbcL transcripts derived from members of the nontarget
group
or from cyanobacteria, but it is possible that they may
be less
discriminating for other chromophyte classes. Apart from
prymnesiophyte
sequences, the closest match to the
E. huxleyi sequence in
the GenBank database is the sequence of another member
of the
Haptophyceae,
Pavlova salina (86% identity). The next most
closely related sequences are all derived from heterokont algae
(77 to
80% identity), which, although they are thought to be more
distantly
related to haptophytes than to cryptomonads or red algae
(
5), include species such as
Pelagomonas
calceolata, which
exhibit extended nucleotide sequence homology
(33-bp identity)
in the 3' region of the gene fragment
analyzed.
When other diatom sequences were excluded, the
rbcL gene of
the raphidophyte
Olisthodiscus luteus (
3) was the
next closest
match (83% identity) to the
S. costatum gene
fragment, and it
had two identical regions (20 and 21 bp) that were
similar in
length to the maximally conserved runs found between the
diatom
probe and the various prymnesiophyte sequences. Whereas the DNA
sequence information and experimental data we have suggest that
cross-hybridization between the diatom probe and other chromophyte
rbcL gene sequences is probably not significant, the
prymnesiophyte
probe probably cross-hybridizes with
rbcL
transcripts derived
from other haptophytes and, perhaps to a lesser
extent, with the
cognate mRNAs produced by members of some other groups
of marine
flagellates. This potential lack of specificity is only
likely
to be a serious practical concern when these motile
phytoplankton
account for a significant fraction of the active biomass.
Empirical
evidence at hand, however, suggests that even under these
circumstances
the prymnesiophyte probe performs
well.
In the mesocosm experiments described here, assorted flagellates
(excluding
E. huxleyi and dinoflagellates) accounted for
a
significant fraction (19 to 23%) of the initial biomass introduced
into the enclosures (
27). During an earlier investigation in
which a general
rbcL gene probe targeting all
microphytoplankton
groups was used (
29), we observed a very
high level of RubisCO
gene expression in both enclosures at zero time
that we now know
was not attributable to either the diatoms or
prymnesiophytes
(Fig.
4 and
5). Since dinoflagellates contributed at
most 0.3%
of the total flagellate biomass, flagellates other than
E. huxleyi were clearly implicated as the source of the high
levels of
rbcL mRNA detected with the general probe at the
very start of the
experiment (c.f. reference
29). In
the present case at least,
therefore, we are reasonably confident that
the prymnesiophyte
probe recognized only the intended target
group.
Temporal variability in rbcL gene expression, primary
production, and development of phytoplankton blooms in
nutrient-stimulated mesocosms.
In agreement with previous
findings (7), the nutrients added to the enclosures
selectively promoted the growth of either diatoms (N/P/Si-supplemented
enclosure) or the prymnesiophyte E. huxleyi
(N/P-supplemented enclosure). The presence of secondary E. huxleyi blooms during the latter half of the experiment was less
expected, but in the N/P/Si-supplemented enclosure this development provided a welcome opportunity to investigate the temporal pattern of
rbcL gene expression in a natural phytoplankton community
undergoing a shift in dominance from diatoms to prymnesiophytes.
Both mesocosms had been filled with nutrient-poor (0.01 µM nitrate,
0.05 µM phosphate, 0.24 µM silicate) postbloom seawater
a day
before nutrients were added at the start of the experiment.
With the
notable exception of phosphate, only very minor changes
in nutrient
concentrations occurred in either of the enclosures
during the first
24 h. The marked stimulation of diatom and prymnesiophyte
rbcL transcription on day 2, however, coincided with
significant
declines in phosphate and nitrate concentrations. Somewhat
surprisingly,
comparatively little (~9% of the starting
concentration) silicate
utilization was evident in the
N/P/Si-supplemented enclosure until
after day 2. However, a very
similar pattern of nutrient assimilation
was observed in another
mesocosm that was supplemented with one-third
(5 µM nitrate, 0.33 µM phosphate, 13 µM silicate) of the concentrations
added to the
first enclosure. In this mesocosm a mixed bloom consisting
of diatoms
and
E. huxleyi also developed during the first week
of the
experiment, but silicate concentrations declined by about
the same
margin in the first 48 h (from 13.23 µM at zero time
to 12.08 µM on day 2), whereas at these lower starting concentrations
nitrate
and phosphate were almost completely exhausted within
the same period
(
27).
The initial preferential utilization of phosphate (and to a lesser
extent nitrate) suggests that the postbloom, diatom-dominated
phytoplankton populations introduced into the enclosures were
probably
not severely Si limited. Consistent with this interpretation,
the
diatom biomass doubled in both nutrient-amended enclosures
during the
first 48 h, although only in the silicate-supplemented
mesocosm
was this growth response sustained beyond the second
day. Addition of N
and P was evidently sufficient to promote
rbcL transcription
in diatoms (and, of course, prymnesiophytes), but
subsequent
translation of this molecular response into an extended
period of
diatom growth and cell division was clearly dependent
on the continued
availability of
silicate.
After day 5, the nutrient concentrations in the mesocosms were
frequently below the level of detection and never exceeded
1 µM
(nitrate), 0.3 µM (silicate), or 0.1 µM (phosphate). These
low-nutrient conditions (particularly the silicate concentration)
clearly restricted further growth of diatoms during the latter
half of
the experiment (
9,
10), but we have made the case
elsewhere
(
29) that the improved light climate which prevailed
after
day 13 may have provided the stimulus for reinitiation of
net growth of
E. huxleyi.
The mean daily irradiance during the second week was 29.4 ± 16.6 mol m
2 day
1, whereas the third week of the
experiment was characterized by
a sustained period of mostly clear,
fine weather (mean daily irradiance,
55.4 ± 10.3 mol
m
2 day
1). However, since very similar
biomass maxima were observed at
the peaks of the primary and secondary
E. huxleyi blooms in the
N/P-supplemented enclosure and the
first of these was associated
with nearly complete exhaustion of the
nutrients added, it is
likely that the development of both secondary
blooms was also
dependent to some extent on the rapid in situ
regeneration of
N and P (
27).
Like the development of the primary blooms, the development of both
secondary blooms of
E. huxleyi was preceded by marked
increases in the amounts of prymnesiophyte
rbcL mRNA.
Significantly,
however, there was little or no evidence that there were
simultaneous
increases in diatom
rbcL gene expression. The
very different molecular
responses exhibited by
E. huxleyi
and the diatoms during the latter
half of the experiment, therefore,
faithfully anticipated the
later growth responses of the two
phytoplankton groups. Some limited
diatom regrowth did occur in the
N/P/Si-amended enclosure after
day 20, but this was when the secondary
bloom of
E. huxleyi was
nearing its peak and some 6 to 7 days after an increase in the
abundance of prymnesiophyte
rbcL mRNA was first
apparent.
Silicate limitation is clearly the most obvious explanation for why
diatoms did not make an appreciable contribution to the
secondary
blooms. Although a convincing case has been made recently
for an active
biological role in this process (
2), it is generally
thought
that silicate remineralization occurs at somewhat lower
rates than the
regeneration of nonsilicate nutrients (
6,
12).
Although we
cannot eliminate the possibility that diatoms may
have been outcompeted
by
E. huxleyi for the low concentrations
of other nutrients
or the possibility that biotic factors such
as preferential grazing or
viral attack had an effect, neither
dissolved nor particulate silicate
accumulated during the latter
half of the experiment. Perhaps not
entirely coincidentally, the
small secondary peak of diatom RubisCO
gene expression in the
N/P/Si-supplemented enclosure was first noted on
the same day
(day 20) that aggregated detritus was pulled into the
water column
following retrieval of lost scientific equipment from the
bottom
of the enclosure. It may also be significant that the silicate
concentration in the fjord water used to replenish the mesocosms
gradually increased from 0.48 ± 0.16 µM (mean ± standard
deviation)
in the 10 days before day 20 to 1.08 and 1.19 µM on days
24 and
25, respectively (
27).
The results presented here are consistent with the premise that the
development of phytoplankton blooms is at least signalled
by, if not
absolutely dependent upon, enhanced RubisCO gene expression.
Increased net production rates were invariably associated with
increases in the abundance of diatom and/or prymnesiophyte
rbcL mRNAs, whereas RubisCO expression was substantially
downregulated
before and between blooms. Why coincident peaks in
prymnesiophyte
and diatom
rbcL mRNA amounts occurred prior
to both primary blooms
requires some explanation, however, since very
different outcomes
in terms of diatom productivity were observed in the
enclosures.
One possible explanation is that the high-level diatom
signal
was due to undetected cross-hybridization between the diatom
probe
and prymnesiophyte
rbcL mRNAs. This possibility can be
effectively
eliminated, however, because the two phytoplankton groups
exhibited
very different molecular and growth responses during the
secondary
blooms. This could have occurred only if the specificities of
the probes were just as discriminating as our initial laboratory
experiments
indicated.
Another possibility is that activation of diatom
rbcL
transcription is silicate independent; however, again, this is somewhat
inconsistent with observations made during the second half of
the
experiment. We have intimated above that the bulk phytoplankton
population introduced into the mesocosms was probably phosphate
limited
rather than silicate limited because (i) diatom biomass
doubled in the
first 48 h in the presence or absence of added
silicate and (ii)
in contrast to P (and N) assimilation, Si assimilation
in the
N/P/Si-supplemented enclosure was significant only from
day 3 onward.
These observations (and the supporting molecular
data) suggest that
diatom RubisCO gene expression can be very
substantially upregulated
provided that silicate is available
to sustain just a single round of
cell division (but probably
not less) and N and P are also
available.
While it is clear that positive changes in the level of RubisCO gene
expression are not an altogether infallible predictor
of phytoplankton
blooms, the use of taxon-specific
rbcL probes
can provide an
early signal and useful indicator of the likely
bloom potential of
individual components of the phytoplankton
community. We used an
rbcL signal normalization procedure that
allowed this
property to be determined only retrospectively, but
Paul and coworkers
introduced the concept of gene expression per
gene dose in which the
abundance of
rbcL mRNA is normalized to
rbcL DNA
(
18,
19,
20). If this approach is taken a stage
further, it
should be possible to determine both variables (
rbcL mRNA
and
rbcL DNA) by quantitative PCR-based techniques that
could
deliver predictive capability in close to real time. Realizing
this potential will depend on developing a much better understanding
of
RubisCO gene diversity in marine phytoplankton, however, so
that
rbcL gene probes and primers can be rationally designed and
their specificity can be
ensured.
In addition to possible applications in coastal zone management and, in
particular, prediction of nuisance blooms, taxon-specific
measurements
of
rbcL mRNA amounts may help us better understand
environmental regulation of carbon fixation in natural populations
of
marine phytoplankton. While the techniques involved are not
trivial,
the target is highly expressed, and the quality of the
information
retrieved gives an instantaneous picture of how a
population is
behaving in situ rather than how it adapts in vitro
during the
prolonged experimental incubations traditionally used
for this
purpose.
| |
ACKNOWLEDGMENTS |
This research was supported by PRIME special topic grant
GST/02/1082 awarded by the Natural Environment Research Council (NERC) of the United Kingdom to M.W. and D.A.P.
We thank the University of Bergen for hospitality at the Espergrend
field station and J. Egge, M. Hordnes, and D. Leslie for managing the
mesocosms and performing the inorganic nutrient analyses.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biological Sciences, University of Stirling, Stirling FK9 4LA, United Kingdom. Phone: (44) 1786 467784. Fax: (44) 1786 464994. E-mail: michael.wyman{at}stir.ac.uk.
Present address: School of Ocean and Earth Sciences, University of
Southampton, Southampton Oceanography Centre, Southampton SO14 3ZH,
United Kingdom.
| |
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Top
Abstract
Introduction
