ICES mar. Sei. Symp., 197: 42-51. 1993

Molecular biology in studies of oceanic primary production

Julie L aR oche , Richard J. G eider, and Paul G. Falkowski

LaRoche, J., Geider, R., and Falkowski, P. G. 1993. Molecular biology in studies of oceanic primary production. - ICES mar. Sei. Symp., 197: 42-51.

Remote sensing and the use of moored in situ instrumentation has greatly facilitated measurements of phytoplankton chlorophyll on global scales with high temporal resolution. However, the interpretation of these measurements with respect to primary production and biogeochemical cycles requires an understanding of physio­logical and biochemical processes in phytoplankton. For example, the use of satellite images of surface chlorophyll to estimate primary production is often based on the functional relationship between photosynthesis and irradiance. A variety of environ­mental factors such as light, temperature, and nutrient availability affect the photosynthesis/irradiance (P vs I) relationship in phytoplankton. Molecular biology provides a means to study the underlying mechanisms by which the primary producers respond to variable environmental factors. We present three examples showing how molecular biology can potentially be used to provide basic insight into the factors controlling primary productivity. The three examples are: (1) Light intensity regu­lation in which an environmental cuc leads to changes in gene expression. (2) Multiple probing of photosynthetic protein expressions to test and explain biophysical and photophysiological responses. (3) Expression of specific proteins induced by nutrient limitation as a potential means of identifying factors limiting photosynthesis in the sea. These examples reflect the personal research interests of the authors, and were selected as illustrations of the potential applications of molecular biology to the study of gene regulation in natural phytoplankton.

Julie LaRoche and Paul Falkowski: Oceanographic and Atmospheric Sciences D iv­ision, Department o f Applied Science, Brookhaven National Laboratory, Upton, N Y 11973, USA; Richard Geider: College o f Marine Studies, University o f Delaware, 700 Pilottown Rd., Lewes, DE 19958-1298, USA.

Introduction

The interdisciplinary nature of oceanography is well exemplified by the many physical, chemical, and biologi­cal principles that are applied to gain understanding of oceanic processes. In the last decade, oceanographers have made use of two new tools to further describe the properties of the oceans: remote sensing allows the study of processes on a global or basin scales, and moored instruments are providing in situ time-series data of coupled biological and physical processes in moving fluids on seasonal time scales. The interpre­tation of the data generated by these new technologies relies on our understanding of basic biological prin­ciples. Throughout the 1970s and 1980s physiological ecologists provided numerous empirical descriptions of the effect of varying environmental factors on the photo­synthetic responses of primary producers (e.g., Platt,

1981). These descriptions have been used to predict integrated water column production (Morel, 1991; Fal­kowski, 1981; Platt et al., 1988); however, in many situations, these empirical relationships do not yield accurate predictions (Campbell and O ’Reilly, 1986; Balch et a i , 1992; Platt et al., 1992).

In order to understand how phytoplankton respond to environmental changes, such as those which are result­ing from global warming and nutrient enrichment, it is not enough to characterize the current distributions of biomass and productivity. It is necessary also to under­stand how phytoplankton biomass, productivity, and species composition are regulated by biological and abiological factors. This knowledge is essential to pre­dict accurately how phytoplankton will respond to alter­ations in the physical/chemical environment. Further­more, the impact of changes in phytoplankton species composition on global biogeochemistry and marine food

ICES mar. Sei. Sym p.. 197 ( 1993) Molecular biology in studies o f oceanic primary production 43

chains can only be predicted if we understand the mech­anisms which control the interactions of individual phytoplankton taxa with their environment. Molecular biology (including protein chemistry and immuno- chemistry) provides new tools with which to investigate metabolic regulation. We feel that molecular techniques can provide new insights into important questions in­cluding, but not limited to, the following. Is the cell division rate of phytoplankton nutrient-limited or is the net population growth rate grazer-controlled? If cell division rate is nutrient-limited, then what is (are) the limiting nutrient(s)? To what extent does light limit primary productivity, and is there an interaction be­tween nutrient limitation and photoadaptation? What environmental factors determine the relative abundance of different taxa at the class (dinoflagellates versus diatoms versus coccolithophorids, etc.) and species levels?

Through the use of molecular biological tools, marine microbial ecologists can address questions concerning the diversity, abundance, and distribution of individual species in mixed microbial populations (Olsen et al., 1986; DeLong, 1992), and a similar approach can be taken to study the diversity, distribution, and abundance of prokaryotic and eukaryotic photosynthetic organ­isms. However, molecular biology and biochemistry can also provide a set of tools and concepts that will improve our understanding of important oceanic processes (reviewed in Falkowski and LaRoche, 1991a). These processes are often empirically described using quanti­tative physiology. Unfortunately, mechanistic models are not available to explain the interaction between the organism and its environment. Here we present three examples of the application of molecular biology to oceanic problems at three different levels of complexity. The examples reflect the interest of the authors and address questions of gene regulation by the environ­ment. While the three examples are neither comprehen­sive nor conclusive, it is hoped that they will serve as models for understanding physiological variations in photosynthetic responses of natural phytoplankton communities. The metabolic flexibility of microbes is well documented. Underlying this flexibility is an ability to regulate the abundance and activity of specific protein responses to changes in the physical/chemical environ­ment. We can examine gene expression at the levels of mRNA and protein abundance, and we can examine the presence or absence of the genes of interest in the DNA of the assemblage under investigation. However, there are fundamental differences in the mechanisms of gene expression between and within prokaryotic and eukary­otic organisms, and qualitatively similar responses to environmental changes may be regulated differently at the genetic level. Therefore, a study of the major taxo­nomic groups is warranted.

Photoacclimation

All phytoplankton studied to date physiologically acclimate to changes in irradiance levels (reviewed in Geider, this issue). Photoacclimation is one of the most important factors determining the functional relation­ship between photosynthesis and irradiance (Falkowski, 1980; Perry et al., 1981) and is evident in the vertical variability of the parameters of the PI curve (Harrison and Platt, 1986), and spectral absorption and chloro­phyll a fluorescence (Neori et al., 1984). This relation­ship, in turn, forms the basis for algorithms used in modelling the spatial and temporal distribution of pri­mary production from satellite images of chlorophyll from the ocean surface (Platt and Sathyendranath, 1988; Balch e ta l., 1992). Despite the numerous models empir­ically describing the relationship between various photo­synthetic parameters and change in irradiance level, we have very little understanding of the molecular and mechanistic basis of this process.

In most algae a change in light intensity is rapidly followed by an inversely correlated change in the cellu­lar amounts of light harvesting chlorophyll proteins (LHCs) (Roman et al., 1988; Sukenik et al., 1988; Falkowski and LaRoche, 1991b). These pigment protein complexes, which bind between 50 and 75% of the total pigments, absorb and transfer excitation energy to the photosynthetic reaction centers. As growth light in­creases, the abundance of the pigment proteins gener­ally decreases and vice versa. The inverse correlation between photosynthetic pigments and light intensity has also been observed in cyanobacteria (Kana and Glibert, 1987) and prochlorophytes (Partensky et a l., 1992). The changes in LHC abundance optimize light collection at low irradiance levels, and minimize photo-oxidative damage at high irradiance levels. In the chlorophyte Dunaliella tertiolecta, the acclimation time scales of different components of the photosynthetic apparatus range from a few hours to a day (Sukenik et a i , 1990). The increase in the LHCs is rapid and is concurrent with an increase in the cab mRNA levels encoding for the LHC apoproteins (LaRoche et al., 1991). A four-fold increase in the mRNA level is detected within 9 h following a transfer to low irradiance. The increase in the cab mRNA following a shift from high to low light is very specific (Fig. 1). There are no detectable changes in the message level of any other photosynthetic gene examined. Interestingly, photoadaptation does not occur during a daily light/dark cycle (Fig. 2), but does occur when a shift in light intensity is superimposed on a light/dark cycle (Postera /., 1984).

Two pathways are involved in the synthesis of the light harvesting complex: chlorophyll biosynthesis and LHC protein synthesis (Fig. 3). Chlorophylls are synthesized in the chloroplast, while LHC is encoded in the nucleus

44 J. LaRoche et al. [CES mar. Sei. Symp., 197 (1993)

High light (steady-state) shifted to low light

at 0 h (36 h)

psaB

chrysophyte Isochrysis galbana (LaRoche et a l., in prep­aration), and the chlorophyte D. tertiolecta (LaRoche et al., 1990). Comparison and analysis of the control re­gions of the cab genes of several species may reveal how light intensity regulates the expression of these genes. The detailed mechanisms leading to the change in pig­ment with light intensity may be different among taxo­nomic groups within the eukaryotes, and between pro­karyotes and eukaryotes, and deserve further study.

Up 4P 4P

Time (h) 0 9 18 27 36

Figure 1. Transcript abundances for several photosynthetic genes as a function of time after a shift from high (700 m -2s ' 1 ) to low light (70 fiE irT 2 s~ ') in D. tertiolecta. Cab encodes the LHC apoproteins, psaB encodes a component of the reaction center from PS1, psbA encodes the D1 protein from PSII; rbcL, the large subunit of Rubisco; and rRNA represent the ribosomal RNÀ. Four micrograms of total RNA per lane were loaded and electrophoresed on denaturing 6% formalde­hyde and 1% agarose gels, transferred to nitrocellulose and probed with 32P labelled DNA fragments containing sequences of the genes of interest. Note the rapid increase in cab transcript after transfer to low light.

and transcribed in the cytoplasm. The low-light induced increase in cab mRNA suggests that the LHC apopro­tein synthesis is probably controlled at the transcrip­tional level; however, experiments with the chlorophyll synthesis inhibitor gabaculine (Mortain-Bertrand et al., 1990; LaRoche et a l , 1991) suggest that chlorophyll biosynthesis is required for LHC protein synthesis to proceed. The initial receptor of a light intensity change has not yet been identified. However, LaRoche et al. (1991) suggested that this response is not directly me­diated by a photoreceptor. They have hypothesized that cab gene expression is repressed in high light by a factor which is regulated via changes in the non-cyclic photo­synthetic electron transport. This has also been sugges­ted to apply to cyanobacteria by Fujita et al. (1987). Genes encoding the major light harvesting chlorophyll binding proteins have been sequenced from the diatom Phaeodactylum tricornutum (Grossman eta l., 1990), the

M odels describing the response o f the

photosynthetic apparatus under various nutrient stresses

The initial slope, a, and maximum photosynthesis rate, , are the major parameters used to describe photo­

synthesis as a function of irradiance. The initial slope of the P-I curve can be related to the maximum quantum efficiency of photosynthesis through the optical absorp­tion cross section. Until recently, the optical absorption cross section (a*) and the quantum yield (r/;m) of marine unicellular algae have been suggested to be constant (e.g., Bannister, 1974; Kiefer and Mitchell, 1983). Although this would simplify the application of bio- optical models, new data show that both (j>m and-a* arë variable (Cleveland et al., 1991; Kolber et al., 1990; Falkowski et al., 1992; Platt et a l., 1992).

Variability in a* arises from interspecific and pheno­typic differences in the ratio of accessory pigments to chlorophyll a, and variations in the extent of intracellu­lar self-shading which can be accounted for by optical considerations alone (Bricaud et al., 1988). The maxi­mum quantum yield is largely independent of irradiance in nutrient replete conditions except for a reduction at high photoinhibitory irradiances. In contrast, there are numerous indications in lab and field data that this parameter varies as a function of limitation by several essential nutrients (Herzig and Falkowski, 1989; Chalup and Laws, 1990; Cleveland e ta l. , 1991; Falkowski et a l., 1991; Greene et al., 1991; Bidigare et al., 1992). Measurements of maximum quantum efficiency, regard­less of how they are obtained, do not provide infor­mation on which nutrient limits photosynthesis nor do they provide insight into the mechanisms by which photosynthesis is impaired under nutrient limitation.

Immunoblotting techniques, combined with biophysi­cal methods, offer a way to determine what components of the photosynthetic apparatus are affected during nutrient limitation (Falkowski, 1992; Greene et al., 1992). We have taken this approach to look at the effects of nitrogen, phosphorus, and iron limitation on the photosynthetic apparatus of P. tricornutum. The time course of cellular chlorophyll and maximal change in

psbA

rbcL

nuclearrRNA

i c e s mar. sd. Symp., 197 (1993) Molecular biology in studies o f oceanic primary production 45

Synchronized light/dark (12 h/12 h) cycle

1.20 200

0.90 •

u 0.60 •• 100 E

cab

Tim« (hrs)

m

psbA ; m m ;

rbcL

nuclearrRNA

Time (h) 12 14 16 18 20 22 24 26 28 30 32 34

Figure 2. Photosynthetic gene expression in cultures synchronized to a light-dark cycle. Cells were grown at 3 5 0 m ~2 s~ ' on alight/dark (12/12) cycle. The dark periods are shaded black on the x axis. Total RNA was isolated from cells every 2 h andabundance of transcripts of cab genes, psbA , rbcL, and rRNA were determined as described in Figure 1

variable fluorescence (Fv/Fm) in this species, following transfer of an exponentially growing culture to media lacking either nitrogen, phosphorus, or iron is shown in Figure 4. The addition of a limiting nutrient after several days demonstrates a capacity for resuming high growth rates and photosynthetic rates within less than a day. The relative abundance of key proteins in the photosyn­thetic apparatus was examined using polyclonal anti­bodies raised against the light harvesting fucoxanthin- chlorophyll-binding proteins (FCP). the reaction center protein (D l) , and ribulose bisphosphate carboxylase oxygenase (Rubisco) (Fig. 5). These three major pro­teins are related to three crucial steps in the photosyn­thetic pathway. The FCP apoproteins are encoded in the nucleus and are involved in light harvesting; D l is

encoded in the chloroplast and is one of four core proteins comprising the reaction center of photosystem II; Rubisco is the first enzyme in the C 0 2 fixation pathway and consists of two subunits, both of which are encoded in the chloroplast genome in chromophyte algae. Nitrogen limitation greatly reduces the abun­dance of the small subunit of Rubisco as well as D 1 in P. tricornutum. The large subunit of Rubisco shows a smaller decline, and the amount of FCP remains rela­tively constant. A decrease in Rubisco and constancy of FCP has also been observed in nitrogen-limited /. gal- bana (Falkowski et al., 1989). In iron-limited cells, the large and small subunits of Rubisco are unaffected while the D l is greatly reduced. Under iron stress, the major light harvesting protein is reduced and may also be

46 J. LaRoche et al. ICES mar. Sei. Symp., 197 (1993)

C e l ls in h i g h l i g h t(Metabolic repression of cob gene expression and chi synthesis via calmodulin?)

\ L o w L i g h t o r

' H ig h L i g h t + DCMU

( D e re p re ss io n ? )

LHCII apoproteins Chlorophyll

o ALA

PROTO

Mg - PME

P Chid

^ c a b T R A N S C R IP T IO N Glu tRNA(Act. D, ru n ‘ on transcrip tion) G lu ta m a te - I - s e m ia ld e h y d e

\J / a m in o t r a n s fe r a s e a c t iv i t y

I( X ) cab mRNA LEVELS ©

(northern blots)

© LHCII TRANSLATION

v T J l h c p i n c o r p o r a t i o n

(thylak. membranes; western blo ts)

Figure 3. Hypothetical model of the regulation of photoadaptation in D. tertiolecta. The model incorporates all the information that is currently available for D. tertiolecta and presents a diagrammatic view of the hypotheses described under Photoacclimation. Question marks indicate pathways for which we presently have no data. (ALA, delta-aminolevulinic acid; GlutRNA, glutamyl tRNA; PRO TO , protoporphyrinIX; Mg-PME, magnesium protoporphyrin IX monoethyl ester; PChld, Protochlorophyllide.

P h a eodac ty lu m tr ico rn u tu m

0.8

0.6a

0.4>

0.2

0.05 6 74320 1

Time (Days)

0.4

M 0.3

0.2

0.075 62 3 40 1

Time (Days)

modified, as suggested by the appearance of an addi­tional higher molecular weight protein. Modification of the light harvesting antenna under iron limitation has been observed in cyanobacteria (Riethman and Sher­man, 1988). The reductions in quantum efficiency (Fv/ Fm) during nutrient limitation are associated with paral­lel decreases in D l , suggesting loss of active PS II reaction centers (Kolber et al., 1988; Greene et al., 1992).

Although it is difficult to generalize our molecular observations to natural phytoplankton communities without looking at a diverse group of algae, biophysical data from natural communities indicate large variations in both quantum yields and absorption cross sections. The variability appears to be non-linearly related to nutrient supply (e.g.. Kolber et a i , 1990). We suggest that detailed studies of the photosynthetic apparatus at the molecular level will help to elucidate the causes of the variations in photosynthetic response in relation to environmental factors. At this stage two simultaneous approaches are needed to attain this goal; (1) We need

Figure 4. Batch cultures of P. tricornutum were grown expo­nentially, and aliquots were transferred to artificial seawater media limiting for nitrogen, phosphorus, or iron. The time- course of nutrient depletion was followed for four days and measurements were made on each day. After four days, an addition of the limiting nutrient to the respective culture (marked by an arrow) showed that the phytoplankton re­covered rapidly from nutrient depiction. Phosphorus limited culture is represented by squares, nitrogen-limited by triangles, and iron-limited by circles.

ICES mar. Sei. Symp., 197 (1993) Molecular biology in studies o f oceanic primary production 47

Phaeodactylum tricornutum

1 2 3 4 +

-ssu

1 2 3 4 +

LSU

_ p ----- Dl

^ F C P

1 2 3 4 5 +

^ I si

-Fe — di

^>FC P****** «s**,™» ~~~-SSU

Figure 5. Western blot of photosynthetic proteins during nutri­ent limitation and recovery in P. tricornutum. Total proteins were prepared as described in LaRoche et al. (1991) and 25 /ug/ lane were electrophoresed on 15% SDS polyacrylamide gels. The protein gels were transferred to nitrocellulose and were sequentially challenged with D l , Rubisco (LSU, large subunit and SSU, small subunit) and FCP (fucoxanthin chlorophyll binding protein) antibodies. The numbers above the lanes represent days after the onset of nutrient starvation and the + signs represent the limiting nutrient addition which occurred after the fourth day in all cases.

to develop a bank of molecular probes that includes antibodies for the various components of the photosyn­thetic apparatus; and (2) we need to improve our under­standing of how each potentially limiting factor alters the expression of photosynthetic genes. An understand­ing at the molecular level will allow us to verify the interpretation of the fluorescence and other physiologi­cal measurements that we routinely perform in the oceans. It will also point out what additional measure­ments need to be developed to obtain an unambiguous picture of the mechanisms prevailing in nature.

Molecular markers indicative o f nutrient

limitation in marine phytoplankton

The vast majority of marine microbes in natural waters are unidentified and not easily cultured. Using molecu­

lar biological techniques, however, it has become poss­ible to identify specific genotypes within natural commu­nities without the need to culture the organisms (Giova- nonni et al., 1990), and within the next decade probes will likely become available for routine identification of marine microorganisms (DeLong, 1992). Similarly, the detection of key genes or proteins in marine organisms can indicate their potential or actual roles in biogeoche­mical cycling [e.g., carbon fixation (Orellana and Perry, 1992), nitrogen fixation (Currin et a i , 1990), methano- genesis or denitrification].

One approach for determining the effect of a particu­lar stress on microorganisms is to seek proteins specifi­cally expressed under the stress condition. This approach has been particularly successful with pro­karyotes, where whole opérons carrying a special func­tion can be derepressed under stress. For example, the phoE operon has been identified using this approach in phosphate starved E. coli, and other examples are listed in Table 1. We have adopted this basic approach to study nutrient stress in eukaryotic algae.

During the course of nutrient starvation in batch culture, we followed protein synthesis by radiocarbon labeling and separated the labeled proteins using sodium-dodecylsulfate, polyacrylamide gel electrophor­esis (SDS-PAGE). In the marine diatom P. tricornutum, a 55 kDa protein is synthesized under phosphorus limi­tation, while iron limitation induces a protein which migrates with an apparent molecular mass of 23 kD (Fig. 6). These stress proteins can also be detected in stained gels and disappear upon addition of the limiting nutri­ent. Using a crude cellular fractionation, we found that small soluble proteins and large membrane proteins are induced by iron or phosphorus limitation in P. tricornu­tum (data not shown) and D. tertiolecta (Fig. 7). We are in the process of characterizing these proteins but we do not yet know their function. However, based on pub­lished information, the large membrane proteins are likely to be components of uptake systems (Reddy etal. , 1988; Scanlan etal., 1989; Harding and Royt, 1990).

Our long-term goal is to obtain a set of molecular probes with which we can determine, using in situ hybridization of preserved samples, whether or not a phytoplankton community is subjected to a particular stress. While universal probes would be the most useful, taxa-specific or even species-specific probes could also be valuable in field studies. However, this approach will require extensive testing to determine the specificity of these probes for a given nutrient stress and the cross­reactivity of antiserum amongst taxa. The identification of the protein function should assist in finding similar indicators in other taxonomic groups when protein structure is not highly conserved. If these goals are attained, in situ hybridization can be coupled with fluor­escence microscopy and flow cytometry for use in field

48 J. LaRoche et al. ICES mar. Sei. Symp.. 197 (1993)

k D a

1 0 6 . 0

Phaeodactylum tricornum0 - 1 2 h 1 2 - 2 4 h 2 4 - 3 6 h 3 6 - 4 8 h

C N P F e C N P F e C N P F e C N P F e

m W

8 0 . 0 ”31

Hf■ p

HP :

4 9 . 5 " Ä 9 À1

3 2 . 5 -

2 7 . 5 -

1 8 . 5 “

Figure 6. Autoradiogram of 14C-bicarbonate labelled protein synthesis after the onset of nutrient starvation induced by the removal of nitrogen, phosphorus, or iron. Exponentially growing cultures of P. tricornutum were transferred to artificial seawater media lacking dissolved inorganic nitrogen, phosphorus, or iron. Protein synthesis was monitored as a function of time after nutrient removal by pulse addition of 50 f i d of 14C bicarbonate followed by a 12 h incubation. The protein samples were run on a 15% SDS-polyacrylamide gel and transferred to nitrocellulose. The results were analyzed by autoradiography. At each time point, C, N, P. Fe represent the control, nitrogen-limited, phosphorus-limited, or iron-limited cells, respectively. The numbers on the left represent the molecular weight of the protein standards. The arrows point to a 55 kDa protein and to the 23 and 21 kDa proteins that are induced under phosphorus and iron limitation, respectively.

Table 1. Induction of specific proteins under nutrient limitation.

Nutrient Organism Protein MW Function Reference

Iron Synechococcus irp A 36 kDa iron acquisition Reddy et a l.,1988

92 kDa membrane Scanlan et al. ,protein 1989

Altermonas 85 kDa Reid andluteoviolaceus 20 kDa Butler, 1991Pseudomonas Fe( III (-regulated Harding andaeruginosa outer membrane Royt, 1990

receptor proteinPhosphorus Pseudomonads PhoE proteins 45 kDa anion Poole and

selection Hancook, 1986channels

E. coli Pit anion Matin et al. ,selection 1989channels

Protogonyaulax phosphatase Boni et al. ,(activity only) 1989

Nitrogen Bacteria glutamine nitrogen Matin et al. ,synthetase (Ntr) metabolism 1989

Sulfur Synechococcus sulfate 33 kDa sulfate Laudenbachpermease transport and Grossman,proteins 1991

Chlamydomonas Arylsulfatase sulfate deHostos et al. ,transport 1987

ICES mar. Sei. Symp., 197 (1993) Molecular biology in studies o f oceanic primary production 49

Dunaliella tertiolecta

so lub le (S) and m em brane (M ) proteins

under nutrient lim itation

kDa

205.0116.0 106.0— 80.0 —49.5 —32.5 — 2 7 . 5 ^

18.5

#tMÉ

S M S M S M S M

- N -P -FeFigure 7. Induction of specific membrane (M) and soluble (S) proteins during nutrient limitation in D. tertiolecta. Four days after the onset of nutrient starvation, D. tertiolecta cultures were labelled with 14C bicarbonate for 12 h. Cells were harvested by centrifugation and, after sonication, total proteins were separated into a soluble and membrane fraction by differential ultracentrifugation. Top arrow points to a 150 kDa protein induced by P limitation, second arrow to a 120 kDa membrane protein induced by Fc limitation, and the bottom arrow to a 23 kDa soluble protein induced by Fe limitation.

studies. However, studies examining alkaline phospha­tase activity as a potential indicator of phosphate limi­tation provide a warning against the blind application of molecular markers in assessing nutrient limitation in the absence of a full understanding of the interactions of the proteins under investigation to other environmental factors and the effects of time lags in physiological responses, and argue for the use of experimental ma­nipulations of natural phytoplankton assemblages under controlled conditions to confirm in situ nutrient status (Wynne and Rhee, 1988; van Boekel and Veldhuis, 1990).

Conclusions

The molecular biological approach, although only re­cently introduced, has already had a significant contri­bution to the elucidation of natural community structure in the ocean. Future advancements in molecular biology may eventually lead to the development of techniques to quantify primary production. We believe that the im­

mediate reward of more widespread application of mol­ecular biological techniques will be in their contribution to a basic mechanistic understanding of how key en­vironmental factors regulate cellular function and gene expression. In this respect, molecular biological studies are a natural extension of physiological ecology which forms the basis of most descriptive models of primary production in the oceans.

A cknow ledgm ents

This research was supported by the US Department of Energy under Contract No. DE-AC02-76CH00016 (JLR, PGF) and by NSF grant OCE 8915084 (RG).

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