Diel rhythms in rubisco gene expression in cyanophytic and chromophytic clades of cultured and natural populations of phytoplankton

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Diel rhythms in rubisco gene expression in cyanophytic and chromophytic clades of cultured and natural populations of phytoplankton

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Title:
Diel rhythms in rubisco gene expression in cyanophytic and chromophytic clades of cultured and natural populations of phytoplankton
Creator:
Brown Kang, Jordan A.
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Tampa, Florida
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University of South Florida
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English
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vi, 64 leaves : ill. ; 29 cm.

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Phytoplankton -- Ecophysiology ( lcsh )
Circadian rhythms ( lcsh )
Gene expression ( lcsh )
Ribulose-Bisphosphate Carboxylase ( lcsh )
Dissertations, Academic -- Marine Science -- Masters -- USF ( FTS )

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Thesis (M.S.)--University of South Florida, 2000. Includes bibliographical references (leaves 28-36).

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University of South Florida
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Universtity of South Florida
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028162150 ( ALEPH )
48097163 ( OCLC )
F51-00149 ( USFLDC DOI )
f51.149 ( USFLDC Handle )

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DIEL RHYTHMS IN RUBISCO GENE EXPRESSION IN CY ANOPHYTIC AND CHROMOPHYT IC CLADES OF CULTURED AND NATURAL POPULATIONS OF PHYTOPLANKTON by JORDAN A. BROWN KANG / A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Marine Science College of Arts and Sciences University of South Florida December 2000 Major Professor: Jolm H Paul Ph D

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Office of Graduate Studies University of South Florida Tampa Florida CERTIFICATE OF APPROVAL Master's Thesis This is to certify that the Master's Thesis of JORDAN A. BROWN KANG with a major in Marine Science has been approved for the thesis requirement on June 15, 2000 for the Master of Science degree Examining Committee: Gabriel A. Var gofl.D.

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LIST OF TABLES LIST OF FIGURES ABSTRACT INTRODUCTION Primary Production C02 Fixation The Calvin Cycle RubisCO Circadian Rhythms TABLE OF CONTENTS MATERIALS AND METHODS San1pling Sites rbcL mRNA Analysis DNA Analysis Phytoplankton Carbon Fixation Cell Co unts Chlorophyll a Analysis Culture and Die! Studies RubisCO Enzyme Activity Pigment Analysis RESULTS C ultur e Studies F i eld Studies DISCUSSION Seward Johnson Lagrangian Study Pelican Lagrangian Study Seward Johnson Vertical Profile Study Pelican Station #2 V e rtical Profile Study Pelican Station #3 Vertical Profile Study REFERENCES AND BIBLIOGRAPHY LEGENDS TO TABLES LEGENDS TO FIGURES 11 lll IV 1 1 3 4 5 8 9 9 9 11 11 12 1 3 1 3 14 15 16 1 6 1 7 18 1 9 20 20 2 1 23 28 37 38

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LIST OF TABLES Table 1 Pelican Lagrangian HPLC Phytoplankton Pigment Analysis 43 11

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LIST OF FIGURES Fig. I Ca l vi n Cycle 44 Fig. 2 Gulf of Mexico Study Site 45 Fig. 3. Norfolk, VA Stud y Site 46 Fig 4 Riboprobe Constructs 47 Fig. 5. PCC 7002 Growth C urv e 4 8 F i g. 6. PCC 7002 mRN A, RuBP an d Carbon Fixat i o n 49 Fig 7 Pavlova Growth C ur ve 50 Fig. 8 Pavlova mRNA RuBP and Carbo n Fixat i on 5 1 F ig. 9a. Sewa rd John so n Lagrangian-Surfac e Drifters 52 F i g. 9b. Seward Jo hnson Lagrangian-15m Drifter s 53 Fig. 10. Seward John s on Vertical Profile 54 F i g. 11. Pel i can Lagrangian mRNA and Carbon Fixa tion 55 F i g. 12. Pe lic a n Lagrangian Autofluorescent Cell Co unt s 56 F ig. 1 3a. P e lican Vertical Profile St 2-mRNA DNA and Carbo n Fixation 57 Fig. 1 3 b Pelican Vertical Profile St 2 Autofl u oresce nt Cell Counts 5 8 Fig. 14a Pelican Vertical Profil e St 2 Cya n o clade pigment s 59 Fig. 1 4b. P elican Vertical Profile St 2 -Chromo clade pigment s 60 Fig. 1 5a. Pelican Vertical Profile St 3 Cyano and Diatom DNA an d C02 F i xa ti o n 6 1 Fig. 1 5b. Pelican Vert ical Profile St 3-Autofluoresce nt Cell Counts 62 Fig. 1 6a. P e lican Vert i ca l Profile St 3 -Cyan o C l ade Pigments 63 Fig 1 6b Pelican Ve rti ca l Profil e St 3 C h romo Clade Pigmen t s 64 Ill

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DIEL RHYTHMS IN RUBISCO GENE EXPRESSION IN CY ANOPHYTIC AND CHROMOPHYTIC CLADES OF CULTURED AND NATURAL POPULATIONS OF PHYTOPLANKTON by JORDAN A. BROWN KANG An Abstract of a thesis submit t ed in partial fulfillment of the requirements for t h e degree of Mas ter of Science Departm e nt of Marine Science Co lle ge of Arts and Sciences Univer s ity of South F l orida December 2000 Major Profe ssor: .Jolm H. Paul Ph.D.

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Where and when different populations of phytoplankton fix carbon dioxide is of major interest in determining the levels of primary production in the oceans. Determining whether a spatial and / or temporal separation in RubisCO activity exists between two major phytoplankton clades, cyanophytes and chromphytes, in open ocean phytoplankton populations would contribute to the knowledge required to determine those levels. It would also contribute to the better understanding of RubisCO-mediated regulation of carbon fixation by cyanophytes and chromophytes. To accomplish this measurements of the levels of RubisCO gene expression were observed between two of the four major clades of Form I rbcL genes during diel experiments in cultures and in open ocean populations. Measurements of RubisCO gene expression were also measured in vertical profiles from open ocean water columns. Additional measurements of RuBP activity, presence of phytoplankton pigments and 14C carbon fixation were made. The findings were used to assess the temporal variation in RubisCO activity during die! experiments and the vertical separation of RubisCO activity between clades of phytoplankton. Analysis of the data revealed that cyanophytic phytoplankton appear to express RubisCO during the morning to early afternoon hours whereas the chromophytic phytoplankton appear to express RubisCO in the late afternoon and evening hours. Cyanophytes also appear to express RubisCO in upper surface waters as opposed to chromophytes that express deeper in the water column. Carbon fixation measurements are greater in the early morning to afternoon hours in cultures of Synechococcus PCC 7002 whereas carbon fixation occurred later in the chromophyte, Pavlova gyrans. In oceanic water columns, cyanophytes were also shown to transcribe rbcL in the upper surface waters while chromophytes expresses rbcL in deeper waters. The detection of phytoplankton-v

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spec ific pi g ments during the s tudy also confirmed the s patial and tempora l distr ibuti on of eyanophytes a nd chromophytes. It see m s apparent that th ere does exist a temporal se p a ration in carbon fixat io n between cyanophytes and chromophytes in l aboratory cultures and in open ocean water columns and a s patial separation in carbon fixation between cyanophytes and chromophytes in open ocean populations based on l eve l s of Rubi sC O ge ne expression. AbstractApproved: ____ _, ____ tv{ajor Pro fessor: John H Paul Ph.D. Pro fessor, Department of Marine Science Date Approved: V I

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INTRODUCTION Primary Production Primary production is the basis for many oceanic life forms. It supports the pelagic and benthic food webs, is influenced by both climate and large-scale ocean circulation, and is a vital link in the biogeochemical cycling of elements Biochemically primary production is termed photosynthesis", and is the conversion of carbon dioxide (C02) and water (H20) into carbohydrates using the energy of light captured by chlorophyll. It consists of two processes : light reactions and dark reactions. The light r eac tion s convert light energy into chemical ene r gy and the dark reactions convert C02 into sugars. These reactions do not require darkness but can take place independently of light. There are three known mechani s m s through which C02 i s convert e d to carbohydrates. The most widespread i s th e 3-carbon pathway (C 3 photosynthesis or the Calvin Cycle). The seco nd method is a 4-carbon pathwa y (C4 photosynthesis) and the third m e thod utilized by xerophytic plants s uch as cacti orchid s, and other succu l ents ha ve a photosynthetic pathway called Crassulacean Acid Metaboli s m (CAM) that fix C02 in the dark as oxaloacetic acid (Smith 1992). Th e key primar y producers in the oceans are the phytoplankton which are pi g mented, photosynthetic microorganisms (always s ingle-celled) and photo sy nthetic bacteria. On a g lobal scale phytoplankton are co n si d ered to b e the most important bioma ss producers in aquatic ecosystems. In the fie l d of biotechnolo gy phytoplankton are becoming potentially important produc e r s of enzymes pharmaceutical s, and nutrac eutica l s which are now produced mainly by gene tically modified microorganisms

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such as yeast and bacteria. They are also involved in bioremediation which involves the biological breakdown and clean-up of environmental pollutants. Phytoplankton populate the top layers of the oceans and freshwater habitats where they receive sufficient solar radiation (photosynthetically available radiation PAR) for photosynthetic processes This top layer of the ocean which is illuminated by sun l ight is referred to as the euphotic zone. In general, the euphotic zone is defined as the area between the sea surface and the depth where light is diminished to 1% of its surface value. The depth of the euphotic zone depends largely on the concentration of organic and inorganic materials dissolved or s uspended in the water column. A large portion of photosynthetic productivity occurs in open oceans mostly b y oxygenic prokaryotes. Phytoplankton fall into a number of categories, including the following: Cyanophytic bacteria (bluegree n algae) which are prokaryote s that perform oxygenic photosynthesis and nitrog e n fixation in some cases. In the oceanic phytoplankton they are represented by Synechococcus sp p and Prochlorococcus (Waterbury 1979 ; Chisholm et al., 1988). Cyanophyticbacteria are quite s mall usually unicellular, though they often grow in colonies and conta in phycobilins. They have the distinction of being the oldest known fossils, mor e than 3.5 billion years old. The oxygen atmosphere that we depend on was ge nerated by numerous cyanophytic bacteria during the Archaen and Proterozoic Eras. The Green algae or Ch l orophyta which include the prasinophytes may be unic e llular mu l ticellu lar colonial or coenocytic (composed of one large cell without cross-walls that may b e uninucleate or multinucleate). They have chlorophyll a and b a s well as seco ndar y pigments and have membrane-bound chloroplasts a nd nuclei 2

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Chromphytic algae compnse three close l y related d i visio n s of algae the Phaeophyta (brow n a l gae) the Bacillariophytes (d i ato m s) and the C hr yso ph yte s (go ld e n algae) The Phaeophyta derive their c h aracter i s tic co l or from t h e l arge amounts of the carotenoid fucoxanthin in their chloroplasts as well as from a n y phaeophycean tannins that might be present. The c hl oro pl as ts also have chlorophyll a, c 1 a nd c2 Chry sop hyte s are si n g l e-celle d fla gellates co n ta inin g th e pigments c hlor op h yll a c and fuc oxant hin Chromophyte s have been found to effectively fix carbon in deeper waters w h ere lig ht availability is low ( Dot y and Oguri 1957 ; Beardall 1 989). T hi s m ay attest t o th e ir competitive advantage for carbon fixat ion in thi s type of environment. C02 Fixation Carbon fixation i s th e pnmary mechanism by which phytoplankton convert at m o s pheric carbon dioxide int o orga ni c ca r bon w hi c h i s u sed as e n e r gy for molecular processe s Thi s removal of atmospheri c carbon dioxide not only i s beneficial for the production of carbohydrates but for the atmosphere it se lf. T h ere ha s been a 25% in creas e in atmo s p h eric carbon dioxide s inc e th e Indu s trial Re v o luti on (Houghton and Woodwell 1 989). Thi s ma y l ead to a n overwa rmin g of the earth's atmosp here whic h h a s repercu s s i o n s o n g l oba l sea l eve l s and ultimately g l oba l climate a theory known as the "gr een h o u s e effec t (Safo n t et al., 199 7; Sc hn e ider, 1 989 1990). The fixation of carbo n is r e g ulated with i n a cycl e known a s t h e Ca l v in Cycle. 3

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The Calvin Cycle ATP and reduced NADP that resulted from the light reactions are used for C02 fixation in a process that is independent of light. C02 fixation involves a number of reactions that is referred to as the Calvin-Benson-Bassham cycle. The Calvin Benson Bassham Reductive Pentose Phosphate Cycle (CBB Cycle) is the primary mechanism in the fixation of carbon by phytoplankton and other carbon assimilating organisms such as aerobic chemoautotrophs and anaerobic phototrophic purple bacteria (Bowien, 1997). It was first di scove red by Melvin Calvin, Alvin Benson and James Bassham in the late 1940's and 50's. With the help o f 14C, and the green algal, Chlorella they were able to identify the intermediate steps that result in the production of carbohydrates Calvin received the Nobel Prize for Chemistry in 1961 for this work (Calvin 1989) The CBB Cycle is a system of 11 reactions involving 13 enzymes (Bassham and Krause 1969 ; Leegood, 1990). All plants and algae remove C02 from their environment and reduce it to carbohydrate by the Calvin Cycle (Fig. 1 ). The process is a sequence of biochemical reactions that reduce carbon and rearrange bond s to produce carbohydrate from C02 molecules. The first step is the addition of C02 to a five-carbon compound (ribulose 1 5-bisphosphate). The six-carbon compound is split, giving two molecules of a three carbon compound (3-phosphoglycerate). This key reaction is catalyzed by Rubisco a large water soluble protein complex. The main energy input in the Calvin Cycle is the phosphorylation by A TP and subsequent reduction by NADPH of the initial three-carbon compound forming a three-carbon sugar triosephosphate Some of the triosephosphate is expo rted from the chloroplast and provides the building block for synthesizing more complex molecules. In a process known as regeneration the Calvin Cycle use s some of 4

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the triosephosphate molecules to synthesize the energy rich ribulose 1 ,5-bisphosphate needed for the initial carboxylation reaction. RubisCO Ribulose-1 5-biphosphate Carboxylase Oxygenase, commonly referred to as RubisCO (EC 4.1.1.39) i s the fir st enzyme in the CBB Cycle It is responsible for up to 95% of the carbon fixation in oceanic phytoplankton (Raven 1995). RubisCO catalyzes the fixation of atmospheric C02 with the five-carbon sugar RuBP to form an unstable six carbon compound which immediately breaks down to form two molecule s of ) phosphogl yce rate (3PGA). RubisCO has a seco ndary activity whereby it can also cataly ze a si milar reaction in which C02 is replaced with 02 to combine with RuBP. In thi s react ion one molecule of )-phosphoglycerate and one molecule of 2 phosphoglycolate are formed (Kellogg and Juliano 1997). This activity is known as the oxygenase function and i s more commonly r efe rred to as photore sp iration. These two reaction s are regulated by RubisCO's specificity factor for C02 or 02 (Omega or Tau) or ratio of carboxy lase activity to oxygenase (Ta bit a 1988). It ha s also been described as th e "effic i e ncy or s ucce ssfu lne ss of C02-dependent growth (Tabita 1999). The probability with which RuBisCO reacts with oxygen vs. with C02 d epe nds on th e relative co ncentration s of the two compounds at the s it e of the reaction In all organisms C02 i s by fa r the preferred s ub strate but as the C02 co ncentration i s ve r y much lower than the oxygen concentration, photorespiration doe s occur at s ignificant le ve l s This s pecificit y appears to be determin e d b y the so urce of the RubisCO enzyme (Jordan and Ogren, 1981 ). The Tau of Cy lind rot he ca Rubi sCO a nd other chromophyticphytic and 5

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rhodophytic enzymes has been found to be high, favoring C02 fixation to oxygenase activity. (Read and Tabita 1992c). Any ability to increase Rubi sCO's s pecifity for C02 would contribute to improving carbon fixation primary production and potentiall y increase crop y ield in agriculture (Read and Tabita 1994; Ramage et al. 1998) Before RubisCO can proceed with these reactions however it must be activated or carbamylated. The first method by which RubisCO is carbamylated involves activation at a specific lysine residue by "activator" C02 RubisCO can be inactiviated by binding cer tain sugar phosphates. These can be removed to restore activity b y RubisCO activase. RubisCO i s found in several forms including Form I as a hexadecamer and Form II as a dimer. Form I is found in bacteria (including cyanophytic bacteria) and in all green plant s (Tabita 1995) and nongreen algae. Fo rm II is found in dinoflagellates and in e ubacteria (Morse et al. 1995; Kello gg and Juliano 1997). The two forms of Rubi sCO h ave different amino acid compositions and kinetics but have active s ites which are s imilar in st ructure (Baker et al., 1977) Form I and II also are different in their s ubstrat e spec ificity and affinity for C02/02 (Tabita 1999) Form I RubisCO has be e n divided into 2 subgroups, g reen (green pl a nt s, gree n a l gae and cyanophytic bacteria) and red (red algae and purple bacteria) and further into 4 clades or subclasses, lA (Bet a and Gamma purple bacteria) IB (cyanophytic bacteria and green algae) IC and ID (diatoms prymnesiophytes, cryptophytes pelagophytes brown and red algae; (Newman and Ca ttolico 1990 ; Dougla s et al., 1990 ; Hwang and Tabita 1991; Valentin and Ze utsche 1990 ; Tabita 1995) There exists also a Form III and a Rubi s CO-like Form IV which a re found in the archaea an d other eu b acte ria ; (Bult et al. 1996 ; Kl enk et al. 1997 ; 6

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Watson and Tabita, 1999) which identifies the ability of such organisms to utilize C02 in a Calvin Cycle-like C02 fixation. However most of these organisms lack phosphoribulose kinase and other essential Calvin Cycle enzymes and have previously been s hown to fix C02 by an altogether separate mechanism (Tabita, 1999) The most common structure of RubisCO (Fig. 5) is composed of large and small subunits, LsSs holoenzyme (Chua and Schmidt, 1978) or more accurately, (L2)4(S4)2 (Knight et al., 1990) with a Mr of about 550,000. The genes encoding the large and small subu nits of RubisCO are rbcL and rbcS respectively. RbcS is nuclear e ncoded while rbcL is encoded in the chloroplast for higher plants and many eukaryotic green algae. In Chromophytic algae, the rbcL!rbcS operon is located in the chloroplast, and IS co transcribed (Berry-Lowe et al. 1982). The s mall subunits do not contribute to the s tructure of the active s ite directly but do influ ence substrate affinities and turnover (Gutteridge, 1991 ) There are a number of inhibitors to RubisCO' s performance. 2' -carboxyarabinitol !-phos phate (2CA 1 P) is a naturally occurring inhibitor that accumulates in the dark and in low-li g ht conditions and which binds to the activated form of RubisCO (Gutteridge et a!., 1986). Another natural inhibitor is a s ubstrat e analogue xyl ulo se-bisphosp hate which affects turnover (Newman and Gutteridge 1994). A third inhibitor i s RubisCO's natural s ubstrate RuBP which accumulates to hig h leve l s in the enzymes inactivated state (Gutteridge and Gatenby, 1995). These inhibitors regulate Rubi sCO activity in vivo Howeve r, there is a lso the importance of tran scr iptional re g ulation which i s often manifested in die! patterns, including circadian rhythms. 7

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<:;:ircadian Rhythms Circadian rhythms, a biological oscillator with a period of about 24 hours were first recognized in leaf movements and were later found to be ubiquitous in eukaryotic organisms (Bunning 1973). It was established in 1960 that living cells use their circadian clocks to adapt to daily changes in their environment (Cold Spring Harbor Symposia on Quantitative Biology, 1960). The life of a plant is dictated by its environment; terrestrial plants, for the most part cannot move and plant cells are minimally protected by an internal environment because each leaf cell must be exposed to ambient air for efficient gas-water exchange and to light for photosynthesis. Under these conditions, a circadian clock is vital to control various phy s iological activitie s such as cell division, pigment ratios and especially those related to photosynthesis (Doty and Oguri 1957) such as oxygen evolution, stomata opening, chloroplast orientation ion or gas uptake and enzyme activities (Sweeney 1987). Circadian clocks improve survival by facilitating adaptation to the environment (Pittendrigh, 1993). To achieve this the circadian clock controls many proce s ses including gene expression (Kondo and lshiura, 1999). The expression of various genes has been shown to be under circadian control such as the chlorophyll alb-binding protein cab2 (Millar and Kay 1997; Lumsden and Millar 1998). 8

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MATERIALS AND METHODS Sa mpling Sites Seawater was collected partly from two cruises one on the RIV Pelican in the G ul f of Mexico (Fig. 2) and the second on th e RIV Seward J o hnson in th e A tlantic Ocean off the coast of Norfolk VA (Fig. 3). Samplin g aboard both shi p s was done using 5L 40L Niskin bottle s attac h ed to a CTD and triggered at the appropr i ate depths around the ch lorophyll a maximum for water column studies. Lag rangian stud i es were also carried out aboard both vessels. Aboard the Pelican we followed a drifter equipped with marker flags and s trobe light s Samples were collected every 4 hours b y nearing the ship to wit h in 1 00 yar d s and lowering t h e CTD to t h e chlorophyll a max depth or lowe rin g a su b mersible pump by han d to 3 m dept h Aboar d the Seward Jolmson we followed two sets of drifters one set at 1 0 m a nd the ot he r set at 25 m depth. The dept h of the coasta l z one determined the depths of the dri fters. Each set of drifters was equipped w ith ARGOS and GPS tracking devices that can te lemeter their position to the sh ip via a VHF radio link. Samples were collected every 12 hours b y n earing the ship to withi n 100 ya rd s of the drifters and lowering t h e CTD array to 10 an d 25 m re s pectively rbcL mRNA Analysis Two hundred fifty milliliters to l L of seawater were collecte d in 2 L plastic s ide arm jugs and/or 20 mLs of cu ltur es ( 1 05 cells mL-1 ) we r e collected in 50 mL screw cap tube s. Both were treated with di e th y lp yrocar bonate (DEPC ; S i gma Chemical Co. St. Louis MO) to 0.1 %, an d filtered in duplic ate on autoclaved 25 mm 0.45 1..1.111 Durapore 9

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polyvinylidene difluoride membranes (Millipore, Inc., Bedford MA). Filters were then placed into sterile 2.2 mL bead beat tubes, label ed and frozen in liquid nitrogen unti l processed in the university laboratory. Bead beat tubes were pre-filled with the following ingredients: 1) 0.5 g baked glass beads (0.1 0.15 mm diameter) 2) 0 .5 mL GIPS extraction reagent. 3) 0.05 mL 2.0 M sodium acetate pH 4.0 4) 0 .5 mL water-saturated phenol and 5) 0.1 mL chloroform:isoamyl alcohol ( 49: 1 ). Tubes were then placed in a mini beadbeater (B i ospec Products Inc.) and processed on high speed for 2 minutes. Tubes were then placed on ice for 15 minutes and centrifuged in a microcentrifuge for 5 minutes to separate the aqueous and organic fractions. The top aqueous phase was recovered and the sample re-extracted twice more with GIPS reagent and 2.0 M sodium acetate. The aqueous phases were combined and precipitated with one volume of isopropanol and 10 flg g l ycogen for 2 hours at -20C. The precipitated RNA extract was microcentrifuged for 10 minutes at 12,000 RPM at 4 C and resuspended in 200 flL of 1.0 mM EDTA, pH 7.0. The RNA was again collected by microcentrifugation, washed 2 times with one vol ume of70% EtOH and r esuspended in 30 flL 1.0 mM EDTA pH 7.0. The RNA was then divided into 3 aliquots, one treated with RNase, one with Dnase and one left untreated. Purified mRNA was then dotted onto Zetaprobe-GT charged nylon filters and either baked in a vacuum oven at 80 C for two hours or UV crosslinked for 12 seconds RubisCO mRNA was detected by probing under stringent conditions (55 C in 50% formamide) using 35Sl abeled rbcL antisense transcripts (Riboprobe constructs Fig. 3) from either the cyanophytic rbcL probe derived from Synechococcu s PCC 6301 or the diatom/chromophytic probe derived from Cylindrotheca sp. N 1 (Pi chard et at., 1996). Dot blots were analyzed using a beta-scanner (BioRad GS363) and quantitated 10

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against standard curves made by in vivo transcription of the rbcL genes from plasmids, w hich were normalized to yeast tRNA. All samples from a cruise or laboratory diel were probed at the same time using standard curves made and tested at one time. DNA Analysis An additional 20 mLs of culture or 500 mLs to 1 L of seawate r was filtered onto 25 mm, 0.45 J.lm Durapore membrane filters which were placed in 2.2 mL screw cap tubes containing 1.0 mL of STE, pH 8.0 (Sambrook et al., 1989) and frozen in a -80 C freezer (for cultures) or liquid N2 until processed in the laboratory. DNA was extracted by adding 1 00 ).lL of 10% SDS and placing the sample in boiling water for 2 min. After cooling cell debris was removed by a 10 min centrifugation at 12 000 RPM followed by collection of the superna t ant. The pellet was re-extracted as above and the combined supernatants were precipitated with 0.1 vo lum e 3.0 M sod ium acetate pH 5.0 and two volumes 1 00% Et hanol at -20 C overnight. The DNA was collected by centrifuging at 12, 000 RPM for 10 min a nd the pellet was h ed wit h one volume of i ce-cold 70% EtOH. The DNA was r esuspended in DEPC-treated s terile deionized water. The DNA extract was purified, dot-blotted and quantified as for mRNA. Phytoplankton Carbon Fixation Rates of carbon fixation were determined by the 1 4C-HC03 teclmique of Strickland and Parsons (1968) as modified by Carpenter and Lively ( 1980). Samples ( 1 0 mLs for cu ltures 325 mLs for natural populations) were placed in steri le 20 mL acid washed liquid sc intillation via l s or 500 mL acid-washed polycarbonate Erlenmeyer fla sks 11

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for cultures and natural populations, respective l y. Light and dark bottle (vials or flasks wrapped with black electrical tape) incubations were used to determine light dependent rates of carbon fixation. Light bott l e values were corrected by subtracting dark bott l e values. Radioactive bicarbonate, e4C]HC03 (53.1 mCi mmor1 ; Amersham Corporation Arlington Heights, I L, USA), was added to each l ight and dark flask at a fina l concentration of 0 5 f.!Ci mr1 A zero time point sample (3 mLs or 100 rnLs) was taken by fil tration through a 25 mm, 0 22 f.!m GS filter (Mill i pore Corp.) and both l ight and dark flasks were incubated under constant cool-white fluorescent lights (average flux: 83 m-2 s-1 ; Sylvania-GTE). Duplicate 3 mL samples were taken after 30 min for cultures or 100 mL samples were taken at two or three hour time points for natural populations and the filters added to glass scintillation vials containing 0.5 mL of 0.5 N HCl and incubated overnight at room temperature. Samp l e radioactivity was determined by dissolving the fil ter in ethyl acetate, adding 10 mL of Ecosint A scintillation fluid (National Diagnostics) and liquid scintillation counting using a Delta 300 model 6891 liquid scintillation counting system (TM Analytic Inc.). Counting efficiency was determined by scintillation counting of a 14C standard (Wang et al., 1975). Sample carbonate alkalinity and total carbon dioxide content were determined by titration with 0.01 N HCl (Strickland & Parsons 1968) Total carbon dioxide and incorporated 14C were used to determine the rate of carbon fixation for each sample. Ce ll Counts Autofluorescent picoplankton were counted as either orange-yellow fluorescing (Synechococcus) or red fluorescing cells (picoeukaryotes and non-phycoerythrin-12

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containing Synechococcus) were enumerated according to the method of Vernet et al. (1990). Samples were filtered onto Irgalan black stained 25 mrn 0.22 11m Nucleopore filters in triplicate. The filters were then placed onto a microscope slide, 8 0 11L glycerol was placed between the filter and the cover slip and the filter stored in the dark and counted onboard the ship within several hours Cells were enumerated using blue light excitation ( 450 nm) and 400X magnification with an Olympus BH-2 epifluorescence microscope For DAPI stained counts of total bacteria 20 mL samples were collected and preserved with filtered formalin to await processing in the laboratory. Cells were s tained with DAPI (1o-5M) and enumerated under UV light using epifluorescence microscopy. Chlorophyll a Analysis Samples for chl a were measured by filtering seawater in triplicate through Whatman GF / F filters and stored at -20 C until further analysis. Filters were extracted with 100 % methanol and chi a was quantified fluorometrically using a Turner Designs fluorometer (Holm-Hansen & Reimann 1978). Cu lture and Diel Studies Two phytoplankton cultures Syn e chococcus sp strain PCC 7002 obtained from the American Type Culture Collection (Rockville MD) and a pryrnnesiophyte Pavlova gyrans CCMP608 obtained from the Provasoli-Guillard Center for Culture of Marine Phytoplankton (West Boothbay Harbor Maine) were grown in batch cultures in SN media and F / 2 medium (Guillard, 1975) respectively in an incubator (Precision Scientific 1 3

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Model 818 Dual Program Illuminated Incubator, Chicago IL) set at 25 C and exposed to a l2h: 12h light: dark cycle using GE fluorescent white bulbs with an average output of 25-50 J.!E/ m2 1.8 L of media were inoculated with cultures growing in exponential phase at a 1 :30 dilution. A diaphragm pump with s terile tygon tubing was used to attach separate acid-washed and sterilized spargers to oxygenate the cultures while sitting atop stir plate s. When cultures were approaching highest cell density as determined by O.D. measurement s, duplicate 20 mL sam ples for each culture were collected for rbcL mRNA and DNA extraction, 10 mL samples were taken for 14C fixation 20 mL samples to measure RuBP enzyme activity 1.0 mL for autofluorescent cell counts and 1.0 mL for spectrophotometric analy s i s at 480 nm. RubisCO Enzyme Activity RubisCO e nzym e activity was mea s ured in toluenized whole cells (Tabita et al. 1978). This involved extracting 20 40 mLs of cells by centrifugation at 20,000 g for 15 min and resuspending in 500 J.!L of a 100 mM MOPS (3-[N-morpholino]-propane s ulfonic acid) KOH buffe r pH 8.0. The san 1ple was th e n treated with 1 /2 volume toluene in a 1.5 mL J.lfuge tube vortexed gently for 3 minutes and allowed to sit in ice for 10 minute s. The toluene la ye r was then removed and the remainin g cells were assayed for RubisCO activity. A I 00 mM MOPS KOH buffer pH 8.0 (buffer A) was added to extracts on ice at samples staggered in ten sec intervals and placed in a 30 C thermal cycler in a hood. At exactly 5 min 100 J.!L ofbuffer A with 50 mM NaHC03 and 25 mM M g Acetate (cold buffer B) and 100 J.!L H14C03 s tock (2 mCi mr1 ) (hot buffer B) was ad de d to eac h extract at sa mple s stagge red at 10 sec intervals. After 5 min 25 fll RuBP 14

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co-factor, 8.0 mM at pH 6.0 to 6.8, or water was added at ten second intervals. Immediately or after 30 min the reaction was stopped with 100 propionic ac id at ten second intervals. The tubes were then bubbled with a pipet (mixed 10 times) and allowed to de-gas in a hood for 60 minutes. After one hour the extracts were centrifuged for ten minutes at low speed. Without disturbing the pellet, 200 was a liquoted into a 20 mL scintillation vial. 10 mL of scintillation cocktail was added to the vial and counted with a liquid scintillation counter. As a further control 10 of hot buffer B was added to 1.0 mL cold buffer B in a tube. 1 00 was taken into a scintillation vial along with 10 mL of scintilla tion cocktail and counted inm1ediately. Pigment Analysis For pigment ana l yses 2 L of water was vacuum filtered ( < 10 psi) through W h atman GF/ F filters in duplicate. The filter s were inm1ediately frozen and stored in l iquid nitrogen until analyzed by high performance liquid chromatography (HPLC) by G.J. Kirkpatrick at Mote Marine Laboratory Sarasota, FL. Filters were extracted in 100% acetone and analyzed using a Hewlett Packard model 10 90 HPLC equipped with a C 18 column and diode array detector as in Millie et a!. (1995). Reverse and forward phase HPLC pigment ana l ysis was performed on the extracts. 15

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RESULTS Culture Studies The following data was collected from a diel study using Synechococcus PCC 7002. Sampling was initiated as cells began to undergo exponential growth. This was estimated b y measuring optical density (O.D.) at 680 nm (Fig 6) The overall growth curve, beginning with inoculation of the study culture volume (1.6 L), shows a linear growth rate (Fig 6). Maximum le vels of mRNA gene expression were observed during mid morning and mid day between the hours of0800 (470 ng L "1 ) and 1200 (450 ng L "1 ) (Fig. 7) The decline in mRNA ge ne expression began s hortl y after the s e hour s w1til the l owest l evel was reached at 2400 (100 ng L"1). RubisCO enzyme act ivity followed a s imilar pattern at least in the beginning of the diel. Highest RubisCO enzyme activity was ob se rved at midday (2.37 and 2.42) and th en a decline until the l owest l evel was r eac h e d at 2400 (0.11 ) whereas the first high est measured value ( 1200) of mRNA coincided with the first high va lu e (1200) of RubisCO enzyme activity, the later p ea k (0800) in mRNA did not coincide with the later peak (12 00) in enzyme act i v ity. Level s of carbon fixation C02 fixe d L"1 ) showed a l a rge increa se at the b eg innin g of the diel around 1600 hours and a much s maller increa se l ater in the diel but a l so a t 1 600 hour s R e l a tin g carbon fixation to mRNA and Rubi sC O enzyme a level s indicat es that unlike mRNA a nd Rubi sCO which showed a co rrelation in incre ases during the die! carbon fixat i on peaked four hours l ater at 1 600 h ours both at the begim1in g a nd at the end of the die I. 1 6

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Figure 8 shows the growt h curve for th e Pavlova gyrans culture. Sampling be ga n at 0800 when cell counts were a little more than 1.4 x 105 cells/mL. Cell counts decreased a s the 12-hour dark period appro ac h ed and incr eased back to 1.6 x I 05 c e lls /mL at 0800 followin g the dark period. Cell co unt s decrea sed for the remaining period of th e diel. Pavlova RubisCO ge ne expression l e vels, as mea s ured b y extra c ted rbcL mRNA rev ea l two distinct increa ses, the first at 1600 h ours (59.94 n g L"1 ) and then a l ess dominant peak a t 1200 hour s (29.97 ng L-1 ) as show n by Fig. 9. Rubi sC O enzyme activ ity levels also s howed two mcr eases, althou g h the in c r eases (1200 to 2000 h ours) and (1200 to 1 6 00 hours ) were n ot as s harp as th e increas es for Rubis CO gene expression. The average of the increa ses however appear to s how t h at RubisCO enzy m e ac tivit y le ve l s were high es t (1.8 a n d 1.0) in the ear l y afte m oo n h ours (1400 to 1600 hours) as expecte d A consis t e nt p a ttern of overall lower values for sample s durin g the se cond h a l f of th e die! was observed for all measured variables. This m ay indicate a reduc e d co ncentr at ion of available nutri e nt s in t h e clo sed c ultur e for cell ac tivit y o r th a t the cells were in s t at ion a r y pha se. Field Studies The n ext s tep in id entify in g di e rh y thm s and vertical pattern s of RubisCO gen e express i o n i s to exam ine phytoplank ton in their n a tur a l e n v ironm ent. T h e followin g data i s presented fro m two cruises o ne a board th e R/V Seward John so n offNorfo lk VA (Fig. 3) a nd the other aboard th e R/V Pelican in th e e a s tern Gulf of Mexico (F ig. 2) T h e fir s t 1 7

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set of observations was derived from Lagrangian studies and the second set of observations was acquired from vertical profile studies performed aboard both cruises. Seward Johnson Lagrangian Study Figure 10 shows the pattern of rbcL expression during a 96 hour Lagrangian drifter study. Samples are from a shallow (1Om depth) pair of drifters. Cyanop h yt ic rbcL expression appears to be greatest, 2.4 4.5 ng r1 in the morning hours, 0758 to 1033, while decreasing and reaching its l owest levels, 0 1.5 ng r1 around 2208 hours. Chromophytic rbcL expression appears to follow an opposite pattern to that of cyanophytic rbcL expression by reaching its highest levels 0.6 1.4 ng r1 in the evening between 2121 to 2208 hours. Not only does ther e seem to be a separate pattern of diel expression between the cyanophytic and chromophytic phytoplankton populations but there also appears to be a difference in th e magnitude of rbcL expression The maximum amount of cyanocphytic rbcL expression (4.5 ng r1 ) is more than three times that of the maximum chromophy tic rbcL. Figure 11 also shows the pattern of rbcL expression during the same 96 hour Lagrangian drifter study but from a deeper set of drifters (25m depth). Cyanophytic rbcL expression appears to follow the same pattern as in the shallow drifters reaching the highest levels 4.0 4.7 ng r1 near mid-morning, 1000 to 1030 hours while decreasing and reaching its l owest levels between 2000 and 2150 hours. Chromop h ytic rbc L expression appears to a l so follow an opposite pattern reaching its highest l evels 3 .0 4.5 ng r1 in the late evening h ours 2 I 00 to 2150, and l owest level s near mid-morning (Fig. 12). 18

PAGE 27

Pelican Lagrangian Study Figures 13 and 14 show the pattern of rbcL expression 14C-fi xatio n bacterial direct counts Chlorophyll a, and orange and red fluorescing cell counts for a 40-hour Lagrangian st udy aboard the RJV Pelican. Cyanophytic rbcL expression appeared to reach its highest levels 130 190 ng r1 around mid-morning, 1000 hours while decreasing and reaching lowe s t levels in the l ate evening and early morning 2200 to 0200 hours. Chromophytic rbcL expression showed only a single peak, SOng r1 at 1800 hours. 14C-fixation seemed to follow the same pattern as cyanophytic rbcL expression, reaching its highest levels 0.3 0.55 u g r1 by mid-morning and decreasing during the evening, 1800 to 2200 hours. Associated with the mid-day increa ses in cyanophytic rbcL expression are increases in orange fluorescing cells (presumably Synechococcus), red fluorescing cells and chlorophyll a. (Fig. 14) Much activity is associated with these same increases in rbcL expression as is shown by chi a increases as well as a small chl a peak at the first 1800 hour mark associated with the only peak in chromophytic rbcL expression. DAPI bacterial direct counts did not s how as clear a rhythm as the other parameters, but did seem to reach its highest levels around mid-day by the end of the study. HPLC pigment analysis (Fig. 15) provided two brief impressions of the phytoplankton populations at the beginning and at the end of the Lagrangian study. This pigment analysis can be u sed to determine algal class abundances in the water under study. At both stations 1 A and 1 K which represent the beginning and the end of the Lagrangian st udy the predominant cyanophytic clade pigments were chlorophyll a2 and 19

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zeaxanthin which are indicators for cyanophytic bacteria prochlorophyte s and chlorophytes. The principal Di ato m clade pi g ment s were chlorophyll s cl and c2 Seward Johnson Vertical Profile Study Figure 1 2 represents a vertica l profile study performed at moorin g station n o. 9 in the DO E s tud y box off t he coast of Norfolk VA, in which both chromophytic and cyanophytic RubisCO mRNA s are compared. Cyanophytic rbcL tran s cription was detected at high level s in the upper s urface waters. C hromophytic rbcL transcription s howed a peak around 20 m and then another at 30m. Pelican Station #2 Vertical Profile Study Figures 16 a and 16b s h ow the di st r i bu tio n of c hloroph yll a, tota l bacterial direct counts oran ge and red fluor esci n g cells, 14C-fixat i o n and rbcL ex pr ess ion fo r a wa t e r co lunm in th e Gulf of Mexico. T h e 45 m depth i s associated with incr eases in o ran ge fluore sc in g cells (phycoerythrinco ntainin g Sy n echococc u s) and DAPI bacterial dir ec t coun t s whi l e the red fluorescing cells d om in ated the deeper 70 m depth The ch lo r ophyll a max was located between the two p opu la ti ons, integrating b o th incr eases at an in t ermediate depth of 58 m The 58 m chlorophyll a m ax a l so co rr e la ted with the carbon fixatio n ma xim um as well as hi g h (5 ng r1 ) cya n op h ytic DNA le ve l s Cyanophytic mRN A leve l s we re hi g h es t o nl y in t h e upp er s urface waters. No chromophytic mRNA or DNA was detected pre swna bl y due to sa mpl e degradation or non -s pecificity of the c hro mop h yt ic probe. Pigment analys i s (Fi gs 18a a nd 18b) s h owed chlorophyll a2 (Prochlo r ococc u s) a n d zeaxa nthin (cyanobacteria) indicative of cya nophytic clade 20

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organisms as well as 19' -Hexanoyloxyfucoxanthin (prymnesiophytes) and chlorophylls c 1 and c2, indicators of diatoms and chromophytic clade organisms, to be the most abundant carotenoids and chlorophylls at the 58 m depth. It is interesting to note that the two most abundant cyanophytic clade pigments were more than twice that (0.50 ug r1 ) of the two most abundant diatom and Chromophytic clade pigments (0.18 ug r1). Cyanophytic mRNA was detected only in the upper s urface waters, suggesting that although the c y anophytic population was present throughout the water column as indicated by cyanophytic DNA lev els, only th e surface population ex hibited RubisCO g ene expression. C02-fixation was modest at the surface, but didn t reach its highest level until deeper in the wa ter column coinciding with the SCM (subsurface chlorophyll maxim um). Pelican Station #3 Vertical Profile Study Figures 20 and 21 show the distribution of Chlorophyll a, total bacterial direct counts, orange and red fluorescing cells 14C-fixa tion and rbcL gene expression for Station 3. The chlorophyll a maximum 85 m is at a deeper depth than station 2 but still a t an intermediate depth between o ran ge fluorescing cells (70 m) and red fluorescing cells ( i 00 m) The red fluorescing cells however, increased at the same depth as chlorophyll a and probably contributed more to chi a than did the orange fluorescing cells DAPI bacterial direct counts were observed at a shallower depth aro und 40-55 m. Carbon fixation was highes t at 85 m coinciding with the chi a max. Cyanophytic DNA howev e r was highest at the s urfac e (20m), but a subsurface increase was seen around 7085 m. Pigment analysis (Figs. 22 a nd 23) show that zeaxanthin, chlorophyll a2 and 2i

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chlorophyll b2 were the most abundant cyanophytic clade pigments around the 70-85 m depth. The most abundant indicators of diatoms which are representatives of the chromophytic clade were 19' -Hexanoyloxyfucoxanthin and 19 Butano y loxyfucoxanthin. They were highest at a slightly deeper depth, between 85 and 100 m Unfortunate l y no mRNA was recovered from this profile. 22

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DISCUSSION This thesis examines the spatial and temporal separation of rbcL gene expression in cyanophytic and cluomophytic clades of phytoplankton in the marine environment as well as the temporal variation in these parameters in lab cultu res, by using group-spec ific RNA probes. Levels of RubisCO activity and the simultaneous measurement of carbon fixing activity in cultured and natural populations of phytoplankton were also measured to assess rhythms in rbcL gene expression and cell activity. Results of the diel studies in cultures and in the field (Pichard et al 1996) revealed that there doe s exist a temporal separation in rbcL gene expression between the cyanophytic and chromophytic clades of phytoplankton The cyanophytic clade ex pres s ing predominantly in the morning to midday while the cluomophytic clade expressing predominantly in the afternoon and tluoughout the evening. Results from the Lagrangian s tudies also show that cyanophytic mRNA tra nscription occurs in th e morning hours to midd ay and is followed by cluomophytic mRNA tran scrip tion in the a fternoon and eve ning hours. Additionally, the Seward Johnson Lagrangian st udy showed that rega r dle ss of depth b e it 10 meters, thi s temporal pattern persists. Since the shallow and deep drifter buoys followed different paths one might qu es tion the spatial separation of RubisCO gene expression between the cyanophytic and Chro mophytic clades. Because o f the shallow depth a stratification o f the water column may not have been pre se nt and the water column was thus a homo geneo u s mixture 23

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Why this temporal separation exists between the clades of phytoplankton may be explained by the availability of nutrients grazing pressure or the recycling of production Nutrient availability decreases in a batch culture with time and in the environment unless there is nutrient influx due to upwelling or recycling, phytoplankton will not have the resources for cellular activity. As there should not be any grazing pressure in a laboratory culture, this applies primarily to the environmental studies. The separation of transcription may be in part a result of grazing pressure on one group of phytoplankton over another, or the type of grazer present at the time. Grazers such as zooplankton may feed on the chromophytic phytoplankton which are at deeper depths during the morning hours and then might migrate upward to feed Whereas at the surface, ciliates would consume the smaller cyanophytic phytoplankton. Vertical profile studies show a spatial distribution between cyanophytic and chromophytic mRNA transcription with cyanophytic activity occurring in the upper water column and chromophytic activity occurring in deeper waters. Phytoplankton pigments can be used for the purpose of assigning phytoplankton to group levels in investigations on phytoplankton group composition. Our pigment data ha s enabled us to determine that the large portion of phytoplankton around the subsurface chlorophyll maximum to b e chromophytic in nature. By analyzing phytoplankton for content and concentration of diagnostic pigments by HPLC, the composition of phytoplankton groups can be determined. The analysis by HPLC is rapid very sensitive, and objective compared to micro scopi c enumerations, which are time consuming and require taxonomic skill. The large amount of data, which can be proces s ed b y HPLC allows the researcher to examine the structure and dynamics of phytoplankton populations. 24

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The study environments utilized in this study are topics of discussion in their own right. The Seward Johnson studies off the coast of Virginia were in a coastal environment near the mouth of the Chesapeake River. Much of the terrigenous inputs such as Nitrate, leads to new production. The Pelican studies were performed predominantly in oligotrophic waters in the Gulf of Mexico. The main source of nitrogen in tlli s enviromnent is ammonia which is recyled in the microbial loop. The studies performed within this project are not without their limitations. Deciphering these distributions and potential rhythms can be variable upon other parameters in the water column and should therefore be included in any future studies. Studies in laboratory cultures attempt to mimic actual environmental conditions however they are not subject to the same forces which nature so carefully applies to the environment. There are certain limitations such as containment or "bottle" artifacts. These prohibit the addition of new nutrient s and the removal of cell debris. Any physical oceanographic feature such as advection irradiance, phytoplankton patchiness and t urbulence is also removed which may provide other requirements for development, e.g. nutrients (Crawford and Purdie 1992). Observations at sea may be hampered by horizontal and vertical transports by migrations by grazing and sinking and also by microdistribution (Sournia, 1974), and by human sampling errors. Phytoplankton have growth and mortality rates on the order of hours to days so their study requires continuous high frequency observations. The application of the Lagrangian study has the advantage over a culture or deck top incubator s tudy (Pichard et al. 1996) in that there are no containment artifacts. However because no tracer was implemented to track the water mass except superficially 25

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with drifter buoys, it cannot be known for certain whether the same water mass was sampled throughout the Lagrangian study. Additionally, no compensation has been made for potential vertical mixing advection or vertical migration of phytoplankton communities. Despite these differences in approach and methodological drawbacks both types of diel studies indicated that some pattern in RubisCO transcription does exist. There are also limitations in the use of gene probes. The group-specific probes are not able to detect all the phytoplankton taxa that fall under the probe's limits For example, dinoflagellates have now been determined to contain an evolutionary diverse fonn II RubisCO (Morse et al., 1995). In addition, while the genetic probes were able to discriminate between evolutionary distinct lineages of RubisCO they are unable to resolve differences in expression between algae within a lineage. This problem has already made itself evident in analysis of phytoplankton-specific pigments where different t axa contain the same pigments (Wright et al., 1991; Millie et al., 1993). The application of phytoplankton pigments to diagnose phytoplankton composition s has been used to predict where and when populations of cyanophytic bacteria or diatoms would b e most abundant in the water column Zeaxanthin and fucoxanthin are two examples that were used to diagnose populations of cyanophytic bacteria and diatoms respectively (Vidussi et al., 2000). The regulation of bio ge ochemically important microbially mediated proces ses at the cellular level is a new field in the growing discipline of molecular ecology. The rbcL gene lend s it self to this typ e of study becau se of its conserved nature, and other genes may not b e as ideally suited to this approach. The RubisCO enzymes facilitate autotrophic g rowth and knowledge of the properties of RubisCO contributes to the 26

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understanding of its evolution, and offe r prospects of modifying and constructing more efficient RubisCO enzymes, those that favor carboxylation over the oxygenase reactions of hi gher plants. The st udy of biogeochemical processes by molecular techniques is a fruitful area for investigation of marine and terrestrial environments. Through such novel and multidi scip linary studies we will improve our understanding of our changing planet. Peop le everywhere must harvest natural resources in order to eat. The world's population is growing by nearly one billion every ten years making it vital to investigate every possible means of sustainable food production both on land and in the ocean. In some parts of the world, the climate or other factors make it impossible to farm the land and in these areas all economic activity and human sett lements are wholly dependent on the ocean Though molecular research has concentrated on improving the photosynthetic processes of organisms it may be an add itional option to increase the organisms' ability to photosynthesize by investigating pathogen resistance or improving the organisms ability to thrive in changing and extreme environments. One pos sibi lity may be to utiliz e the chromophytic rbcL gene which may be more adept in a light-limited environment to fix carbon By genetically modifying a terrestrial plant's carbon fixing ability it may become more efficient at fixing carbon. However one would have to take into consideration that if you increase carbon fixing capability in a terrestrial plant, it would also require more nitrogen unless the plant can employ a more efficient nitrogen fixing sys tem Which in turn can be altered by utilizing nitrogen fixing genes from certain cyanophytic bacteria. 27

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REFERENCES AND BIBLIOGRAPHY Akazawa, T. Takabe, T., Kobayashi, H (1979): Molecular Evolution of RibuloseI ,5Bispho s phate Carboxylase/Oxygenase (RuBisCO). Trends Biochem. Sci. 9, 380-383 Ambulos, N P. Jr., Duvall, E.J., Lovett, P.S. (1987): Method for blot-hybridization of mRNA molecules from Bacillus sub til is. Gene 51, 281-286. Bainbridge G (1995): Engineering Rubisco to change its catalytic properties. J Exp. Botany 46 1269-1276 Baker S.H., Jin S., Aldrish H .C ., Howard, G.T., and Shively, J.M. (1977): Insertion mutation of the form I cbbL gene encoding ribulose bisphosphate carboxy la se/oxygenase (RubisCO) in Thiobacillus neapolitanus result s in expression of form II Rubisco loss of carboxysomes, and an increased C02 requirement for growth. J Bacterial 180 41334139. Bassham J.A., and Krause G.H. (1969): Free energy changes and metabolic regulation in steady state photosynthetic carbon reduction. Biochim Biophys Acta 189, 207-221. Beardall J. & Morris I. (1989): The concept of light intensity adaptation in marine phytoplankton: some experiments with Pha eodactylum tri co rnulum Mar Bio 37 377387. Berry-Lowe S.L., McKnight T.D., Shah D.M. and Meagher R.B. (1982): The nucleotide se quence expres s ion and evolution of one member of a multigene family e ncoding the small subunit of ribulo se -I 5-bisphosphate carboxylase in soy bean. J Mol Appl Gene t I 483-498 Bowien, B. and Mayer, F. (1997): Further s tudie s on the quaternary structure of d ribulo se1 ,5-b i s phosphate carboxylase from Alcaligenes eutrophus. E ur J Biochem 88 97-107. Branden C.-I. Lindqvist Y. and Schneider G. (1991): Protein engineering ofRubisco, Acta Crys t sec t B47 824-835. Bult, C.J. White, 0., Ol se n G.J. et al (1996): Complete genomic sequence of the methano ge nic archeon, M eth anococcus jannaschii. Arch Microbio/1 09 15-19. Bmming E. ( 1 973): The Physio l ogical clock (3rd ed) Springer Verlag. Calvin, M. ( 1989) : Forty years of photosynthesis and related activities. Photosyn R es 2 1 3-16. 28

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Golden S.S ., Bru ss lan J. and Ha se lkorn R. (1997): Genetic engineering of th e cyanobacterial chromosome. Methods Enzymol 153, 2 15-231. Grobbe lar N., Huang, T.-C., Lin H .Y., and Chow, T.J (1986): Dinitrogen-fixing Endogenous rhythm in Synechococcus RF -1. FEMS Microbial L e tt 3 7 173-1 77. Guillard R.R.L. (1975): Culture of phytoplankton fo r feedi n g m a rine invertebrates. In: Smith, W.L. a nd Chanley M.H., (Ed) Cu lture of Marine Inver tebra t e Animals. Plenum Press NY 26-60. G utt e rid ge, S (1991 ): The relative ca tal yt ic s pecificities of the l arge subunit core of Synechoccus ribulose bi s pho s phate carboxylase / oxygenase J Bioi Chern 266, 73597362. Gutteridge S. Parry M A.J. Burton S. Ke ys, A.J. Mudd A., Feeney, J. Servaites J. C and Pi erce, J. (1986): A nocturn a l inhibitor of carboxylation in le aves. Nature 324, 27 42 76 G utterid ge, S. and Gatenb y,A .A. (199 5) : Rubi sCO sy nthe sis, assembly me chan i s m and r eg ul ation. The Plant Cell7, 809 -8 19. Gutte rid ge, S. Newman J., Herrm a nn C., and Rho ades, D. (1995): The crysta l structur es of Rubisco and opportunities for manipulatin g photo sy nthe sis. J. Ex p. Bota ny 46:10551060. Hartman F.C. Harpe l M .R. (1994): Structure, Function, R eg ul a tion and Assemb l y of D-Ribo se-1, 5-Bisphosphate Carboxylase/Oxygenase. Ann R ev Biochem 63 1 97-234. Holm-Han se n 0., and Riem a nn B (1978) : C hl orop h y ll a determination : improvements in m e thod o l ogy. Oikos 30 438-447. Houghton, R.A. a nd W oodwe ll G.M. (19 89): Globa l climatic change. Scien t Am 260, 36-44. Huang, T.-C., and Grobbe l aar, N (1995): The C ir ca dian C l ock m t he Prokaryo te Synechococcus RF-1. Microbio/14 1 535-540. Huang, T. -C., Tu J C how T .-J. a nd Chen T.-H. (1990): Circa dian Rhythm of th e Prokaryote Synech o coccus s p RF -1. Plant Physio/92 531-533. Hwang S.R. and Tabita, F .R. (1991 ): Co tr a n sc ripti o n d e du ce d primar y s tructure and express ion of the c hloropl ast-e n co d ed rbcL and rb cS ge n es of the marine diatom Cylind roth ec a sp. S train Nl. J Bioi C h em 266 6271-6279. J ordan, D.B and Ogren W.L. (1981): Speci es variation in th e s pecific ity of ribu l ose bi s phosph ate carboxylase/oxyge n ase Na tur e 291, 5 1 3 5 1 5. 3 0

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Jordan, D B., & Ogren, W.L. (1983): Species variation in kinetic properties of ribulose-! carboxylase / oxygenase Arch Biochem Biophys 227, 425-433. Kellogg E., and Juliano N.D. (1997): The structure and function of RubisCO and their implications for sys tematic studies. Amer J Botany 84(3) 413-428. Knight, S., Andersson, 1., and Branden, C.-I. (1990): Crystallographic analysis of ribulose 1 ,5-bisphosphate carboxylase from spinac h at 2.4C resolution. J Mol Bioi 215, 1 1 3 -160 Kondo T. and Ishiura M. (1999): The circadian clocks of plants and cyanobacteria. Science 266, 1 233-1236. Leegood, R.C (1990): Enzymes of the Calvin cycle. In: Lea P .A. (Ed.) Methods Plant Biochem Academic Press London, 3 15-37 Legendre L., Demers, S., Garside C., Haugen, E.M. Phinney D.A. Shapiro, L.P. Therriau lt J.C. Yentsch C.M. (1988): Circadian photosynthetic activity of natural marine phytoplankton isolated in a tank. 1. Plankton Res. 10: 1-6. Lorimer, G. H., Badger M. R., Andrews T. 1. (1983): D-Ribo se-1,5-Bisp hosphate Carboxylase/Oxyge nase improved methods for the activation and assay of catalytic activities. Annal Biochem 78, 66-75. Lum s den P.J. and Millar A.J. eds (1998): Biological Rh ythms and Photope riodism in Plants. BIOS Scientific Publisher Ltd. Lundqvi s t T and Schneider G. (1989) : Crysta l struc ture of the complex of ribulose 1 5bisphosphate carboxylase and a transition state analogue 2-carboxylase-D-arabinitol I 5 bispho s phate. J Bioi Chern 264 7078-7083. McFadden, B.A. (1980): A perspective of Ribulose Bisphosphate Carboxylase/Oxyg enase th e Key Cata ly st in Photosynthesis and Respiration. Ace Chern Res 13, 394-399. Millar A.J. and Kay, S.A. (1997): The genetics of phototransduction and circadian rhythms in Arabidopsis. Bio Essays 19, 209-214. Millie D.R. Paerl H.W. and Hurley J P (I 993): Microalgal pigment assessments u sing high-performance liquid chromatography: a sy nopsis of organismal and ecological a pplications Can J Fish Aquat Sci 50 2513-2527 Millie D.F., Kirkpatrick, G.J., Vinyard B.T. (I 995): Relating photosynthetic pigments and in vivo optical densit y spectra to irradiance for the Florida red-tide dinoflagellate Gy mnodinium breve. Mar Ecol Prog Ser I 20 65-75. 31

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Miziorko, H.M., and Lorimer G.H. (I 983): Ribu l ose-1 5Bispho s phat e Carboxy l ase Oxygenase Ann Rev Biochem 52, 507-535 Morse D., Fritz, L. and Hastings, J.W (1990): What is the C lock ? Translational Regulations of Circadian Biolumin esce nce. Trends in Biochemical Sciences 15, 262 265. Morse D. Salois, P., Markovi c, P ., Hastings, J.W (1995): A nuclear encoded for m II RubisCO in dinoflage ll ates. Science 268, 162 2 -1624. Newman S.M., and Cattolico R.A. ( 1 990): Ribulose bisphosphat e carboxy la se in a l gae: Synthesis, enzymology and evolution. Photosynth R es 26 69 85. Newman J and Gutteridge, S ( 1 994): St ru cture of an effecto r indu ce d in activate d state of ribulos e 1 5 -bisph osp h a te carboxylase / ox y ge n ase: The binary complex b etwee n e n zy m e and xy lulo se 1 ,5 -bispho sp h ate Struc tu re 2, 495-502. Palenik, B. & Haselkom R. (1992): Multiple evo lutionar y origin s of prochlorophytes t h e ch l orop h yll b-containin g prok aryotes. Nature 355 265-267. Palmer, J.D. (1996): Rubisc o surprise s in dinoflagellates. The Plant Cell8, 3 43-345. Paul J.H. and Picha r d S.L. (1995): Ex tr ac ti on of DNA and RNA f rom Aquatic Environ m e nt s In: Nucleic Acids in the Envi ronment. (Eds: Trevors JD ; Van E ls as JD ) Springe r Verlag, Berlin 153177 Paul J.H. Pichard S.L., Kang J.B Watson G.M.F. and Tabita, F R ( 1 999) : Evid e nce for a clade-specific temporal a nd spatia l separation in ribulose bisphosphate carbox y l ase gene express ion in phytop lankt o n populations off Ca pe Hatter as and Bermuda. Limnol O c eanogr 44 12-23. Pau l J.H., A lfr e ider A., Kang J.B. Stokes R .A., Griffin D. Campbell, L. and Orno l fsdottir, E. (1999): Form lA rbcL Transcripts Associated With a Low Salinity / High C hloroph yll Plume ("Green River ) In The Eas t e rn Gulf of M exico. In Press. P au l J.H. Kang, J.B. and Tabita F.R. (2 000 ): Die! patterns of regulation of rbcL transcription in a cyanobacterium and a p ry mn es ioph y te Mar Bi otech (In Press) Pichard S.L., P a ul J H (1991): D etection of Gene Ex p ress ion in Genetically Engi n ee r e d Microorganisms and Natural Ph y t oplank t o n Populations in th e M arine E nvironment b y mRNA A naly sis Appl Environ Microbial 5 1 1721-17 27. Pichard S.L. Fr i sc her,M. E., Paul J.H ( 1 993): Ribulose Bisphosphate Carboxy l ase Gene Exp r ess i o n in Subtropical Marine Phytoplankton P o pulations. Mar Ecol Prog Ser I 01, 55 65. 32

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Pichard,S.L., Brown, J.B., Campbell,L., Tabita F.R., Paul,J.H. ( 1996): Regulation of Ribulose Bisphosphate Carboxylase Gene Expression in Natural Phytoplankton Communities. I) Diel Rhythms. Mar Ecol Prog Ser 139 257-265. Pichard,S.L., Campbell,L., Carder,K. Kang, J.B., Patch,J ., Paul,J.H. (1996): Regulation of Ribulose Bisphosphate Carboxylase Gene Expression in Natural Phytoplankton Communities. II) Variation in Size Fractions and Through the Euphotic Zone. Mar Ecol Prog Ser. (Submitted) Pichard,S.L. Campbell,L., Carder K., Kang, J.B. Patch,J., Tabita, F.R. and Paul, J.H. (1997): Analysis of rib ulose bisphosphate carboxylase gene expression in natural phytoplankton populations by group-specific gene probing. Mar Ecol Prog Ser 149, 239253. Pichard,S.L. Campbell,L. Paul,J.H. (1997): Diversity of the Ribulose Bisphosphate Carboxylase/oxygenase Form I Gene (rbcL) in Natural Phytoplankton Communities. (In Review). Pittendrigh, C.S. (1993): Temporal organization: reflections of a Darwinian clock watcher. Ann Rev Physiol 55, 17-54. Puiseux-Dao S. (1981): Ce ll-C ycle Events in Unicellular Algae. Can Bull Fish Aquatic Sci 210, 130-149. Putt,M., Rivkin,R.B., Prezelin,B B. (1988): Effects of altered photic regimes on diel patterns of species-specific photosynthesis. I. Comparison of polar and temperate phytoplankton Mar Bio/97, 435-443. Ramage R.T., Read, B.A. and Tabita F.R. (1998): Alteration of the alpha helix region of cyanobacterial ribulose 1,5-bisphosphate carboxylase/oxygenase to reflect sequences found in high substrate specificity enzymes. Arch Biochem Biophys 349, 81-88. Raven J.A. (1995): Inorganic carbon assimilation by marine biota. J Exper Mar Bioi Eco/203, 39-47. Read, B.A. and Tabita, F .R. (1992): A hybrid ribulose bisphosphate carboxylase / oxygenase enzyme exhibiting a substantial increase in substrate specificity factor. Biochem 31, 5553-5560. Read, B.A. and Tabita F .R. (1994 ): High substrate specificity factor ribulose bisphosphate carboxylase / oxygenase from eukaryotic marine algae and properties of recombinant cyanobacterial Rubisco containing "al ga l residue modifications. Arch Biochem Biophys 312 210-218. Reid C.D. Fiscus, E L. Burkey K.O. (1998): Combined effects of chronic ozone and elevated C02 on Rubisco activity and leaf components in soybean Glycine max. 33

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Vidussi F., Marty J.C., Chiaverini, J. (2000): Phytoplankton pigment variations during the transition from spring bloom to oligotrophy in the northwestern Mediterranean Sea .Deep Sea Research I 47, 423-445. Wang D.H. Willis D .L., Loveland, W.D. (1975): Radiotracer methodology in the biological, environmental, and physical sciences. Prentice-Hall Englewood Cliffs NJ. Waterbury J B., Watson, S.W., Guillard, R.L. and Brand, L.E. (1979): Widespread occurrence of a unicellular marine, planktonic cyanobacter ium. Nature 277, 293-294. Watson, G.M.F. Tabita F.R. (1995): Organization and Regulation of Carbon Fixation Genes in the Marine Cyanobacterium, Synechococcus sp. WH7803. Department of Microbiology. The Ohio State University. Watson, G.M.F., and Tabita F.R. (1996): Regulation, unique gene organization and unusual primary structure of carbon fixation genes from marine phycoerythrin containing cyanobacterium Plant Mol Biol32, 1103-1115. Watson, G.M.F., and Tabita F.R. (1999) : Unusua l ribulo se 1 5-bisphosphate carboxylase / oxygenase of anoxic Archaea. J Bac t e riol181 1569-1575. Wright S .W., Jeffrey S.W. Mantoura R.F.C. Llewellyn C.A. Bjornland T. Repeta D. and Wel sc hmeyer N. (1991): Impro ved HPLC method for the analysis of chlorophy ll s and carotenoids from marine ph y toplankton. Mar Ecol Pro g Ser 77 183 196. 36

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LEGENDS TOT ABLES Table 1. Phytoplankton pigment analysis from R/V Pelican Lagrangian Study. Using HPLC pigment analysis was performed at the beginning (Station A) and at the end (Station 1K) of a Lagrangian Study. Chlorophyll a2 and Zeaxanthin were the most predominant Cyanophytic clade pigments while chlorophy lis c 1 c2 and 19'Hexanoylofucoxanthin were the most predominant Chromophytic clade pigments. 37

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LEGENDS TO FIGURES Figure I. Diagram of the role that RubisCO has in the overall picture of C-fixation RubisCO is transcribed and translated depending on whet her it is the large or small subunit. It is then incorporated into the Calv in Cycle as the I st step in carbon dioxide fixation. It inc orporates C02 into organic form 3-phosphoglycerate which i s then incorporated into the TCA Cycle where it is assimilated into amino acids and proteins. Rubisco also has an oxygenase activity which is not shown here. Figure 2. Map of Gulf of Mexico study site and location of stations sampled. Not all stations are represented in this thesis. Stations 1 B through I K were open ocean stations during the Lagrangian Study. Stations 2 and 3 were also an open ocean stations for a Vertical Profile Studies. Figure 3. Map of the Atlantic Ocean study s ite east of Norfolk VA. Sampling for the Lagrangian and Vertical Profile Studies a ll took place within the DOE box Figure 4. Gene constructs showing plasmids for cyanophytic and chromophytic rbcL mRNA probes The specificity of the co n sructs depends on the promoter and the enzyme used at the multiple cloning site near the gene of interest. Figure 5 Growth curve of Syn ec ho cocc us spp PCC 7002 cells / rnL, during laboratory di e l study. Sampling began at 0800 hours while cells were dividing. A stationary phase is seen during the evening period, 1800 to 3000 hours into the diel and then. Figure 7 Levels ofmRNA, RubisCO activity and carbon fixation for Syn ec ho coccus spp PCC 7002 during diel study. Figure 8 Growth curve ofPavl ova gyrans during lab oratory die! study. Sampling began at 0800 hours as cell growth a ppear s to be decreasing for the evening hours ( 1800 to 38

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0600). Cell growth then increases in the early morning 0800 hours then experiencing a rapid death phase for the remaining of the diel. Figure 9. Levels of mRNA, RubisCO enzyme activity and carbon fixation for Pavlova gyrans during die! study. mRNA (open circles) carbon fixation (closed squares) and RubisCO enzyme activity (closed triangle) were all at their maximum levels between 1200 to 1600 hours at the beginning of the die! study. (All measurements decreased during the evening period (black bar). Only mRNA and RubisCO enzyme levels shows another peak between 1200 to 1600 hours after the evening period.) discussion Figure 10. Lagrangian Study aboard the RIV Seward Johnson Data represents cyanophyticphyte (open squares) and chromophyticphyte (closed circles) mRNA for the shallow drifters. C y anophyticphyte mRNA is detected at its highest levels during the mid-morning hours while chromophyticphyte mRNA is detected at its highest levels during the evening hours. Figure 11. Lagrangian Study aboard the R/V Seward Jolmson. Data represents cyanophyticphyte (open squares) and chromophyticphyte (closed circles) mRNA for the deep drifters. Cyanophyticphyte mRNA is again detected at its highest levels during the mid-morning hours while chromophyticphyte mRNA is again detected at its highest levels during the evening hours. Figure 12 Vertical Profile Study aboard the R!V Seward Jolmson at mooring station 9. Cyanophyticphyte mRNA (closed squares) is detected at its highest levels in the upper water column and decreasing with increasing depth. Chromophyticphyte mRNA (pluses) is detected at moderate levels in the upper water colunm then decreasing with increasing depth and then showing an increase past 25 m depth. 39

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Figure 13. Lagrangian Study aboard R/V Pelican showing Cyanophytic and Chromophytic mRNA and carbon fixation Cyanophytic mRNA (open squares) and carbon fixation (closed diamond) are detected at their highest levels during the mid morning hours (1000 to 1400 hours) throughout the study Chromophytic mRNA (closed circles) is detected only in the beginning of the study showing a peak at 1800 hours. Figure 14. Lagrangian Study aboard R/V Pelican showing autofluorescent cell counts and levels of chlorophyll a Orange fluorescing cells (open triangles) and red fluorescing cells (x's) are detected at their highest levels during the mid-morning hours (1000 to 1400 hours) throughout the study. Bacterial direct counts (BDC, open diamonds) are detected at moderate levels throughout the diel except for a peak at the end of the study at 1 000 hours. Chlorophyll a (closed squares) maximums were detected in the morning hours along with Orange and Red fluorescing cells throughout the study. An additional peak was detected just prior to the first evening period between 1800 to 2200 hours. Figure 15. Station 2, Vertical Profile Study from RIV Pelican. Cyanophytic mRNA (open squares) was highest at the surface 15 m, and decreased with increased depth Cyanophytic DNA (closed triangles) was moderate at the surface and reached maximum levels at 58m which coincided with the maximum value for net carbon fixation (open diamond) which was moderate at the surface and decreased after 58 m. Figure 16. Autofluorescent cell counts from station 2, Vertical Profile Study from RIV Pelican Orange fluorescing cells (closed triangles) and bacterial direct counts (open diamonds) are prominent in the surface waters and reach their maximum levels at 45 m Chlorophyll a (open squares) and Red fluorescent cells (x' s) are low in the surface waters and show increases at 58 m and 70 m respectively. 40

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Figure 17. Cyanophytic clade pigments from station 2 Vertical Profile Study from RN Pelican. Chlorophyll a2 (closed diamonds), Zeaxanthin (asterisk) and chlorophyll b2 (closed squares) are detected at their highest levels between 58 m and 70 m Figure 18. Diatom clade pigments from station 2, Vertical Profile Study from R/V Pelican. Chlorophylls c1, c2, (closed diamonds) 19'-hexanoyloxyfucoxanthin (closed triangles) Fucoxanthin (closed squares) and 19 -Butanoyloxyfucoxanthin (asterisks) are detected at their maximum levels between 58 m and 70 m. Figure 19 DNA and carbon fixation from station 3 Vertical Profile Study from RN Pelican. Cyanophytic DNA (closed squares) was detected at its highest level at 20 m then at moderate levels with increasing depth Another peak is seen between 70 m and 85 m. Chromophytic DNA (open triangles) was detected at the surface and then decreased with increased depth Carbon fixation (open diamonds) was detected mostly at the surface with a peak at 85 m. Figure 20. Autofluorescent cell counts from station 3, Vertical Profile Study from RN Pelican. Bacterial direct counts (closed diamonds) are prominent in the surface waters and r eac hed maximum levels between 40 m and 55 m Chlorophyll a (closed s quares) red fluorescent cells (x's), and Orange fluore scing cells (open triangles) are low in the surface waters and show increases between 70 m and 1 00 m depth. Figure 21. Cyanophytic Clade pigments from station 3 Vertical Profile Study from R/V Pelican Zeaxa nthin (x's) was detected prominently in the surface water colum until I 00 m depth with a peak at 70 m. Chlorophyll a2 (closed diamonds), and chlorophyll b2 (ope n squares) were detected at their highest lev e l s between 85 m and I 00 m 41

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Figure 22. Diatom Clade pigments from station 3, Vertical Profile Study from RIV :Pelican Chlorophylls cl, c2, (open diamonds) 19'-hexanoyloxyfucoxanthin (closed triangles) Fucoxanthin (open sq u ares) and 19' -Butanoyloxyfucoxanthin (aseterisks) are detected at their maximum le vels between 85 m and 1 00 m. 42

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Table 1 Pelican Lagrangian HPLC Phy toplankton Pigment Analysis Chlorophylls (J.lg/L) and Carotenoids (J.lg/L) ; yanopJ lYilC a e 11 ments C h f Cl d p c N N .,.... c :.c ro ..a ..a c ...... ..c c c c ..c c ro 0 Q) X E Q. Q. Q. . 1A 06 0 .064 0 003 0.005 0 .000 0 .061 0 .001 0 .000 1K 22 0 078 0 004 0 .006 0 000 0 068 0 001 0 .001 Ch h Cl d p romoplvtlc a e 1gments s c ..c ..c N c c (.) ro ro X c X c 0 c 0 S .,.... (.) ..c (.) (.) ..c :::::1 ..c :::::1 ..c c c --0 Q) (/) c ro c c E ro X X ro X ro !1l X 0 0 X 0 X U5 i= Q. 0 c 0 0 (.) ...... c 0 "0 !1l 0 0 :::::1 c !1l i5 c i5 .._ u.. .2 ro i5 ro X 5 ..c Q) () I al ' b> b> .,.... .,.... 1A 06 0 015 0 004 0 017 0 .00 3 0 000 0 .00 3 0 000 1K 22 0 012 0 .005 0.021 0 .005 0 000 0 003 0 000 43

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Fig. 1 Calvin Cycle DNA rbcLrbcS RNA pol transcription () mRNA ribosome Calvin t translation 0 Cycle ubisco Protein ( 0 TCA g Cycle Amino acids Proteins ()0 0 0 44

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Fig. 2 Gulf of Mexico Study Site 84" 82" 45

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Fig. 3 Norfolk, VA Study Site Chesapeake Bay Atlantic Ocean DOE Box 37 0 Virginia D 36 46

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Fig. 4 Riboprobe Constructs Synechococcus sp. PCC6301 rbcL {Cyano) Cylindrotheca sp. lt>cL (diatom) M>pA T7 transcription I SpG transcript ion I ted : Sense (S) RNA probe 0 RNA polymerase promoters Multiple cloning site ffi Cloned sene 47 ;;r Sp6 Antiser. se {AS) ANA probe

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Fig 5 PCC 7002 Growth C u rve 1 60E+06 1.40E+06 1 20E+06 1 00E+06 _. E 8 0 0E+05 .!!l (jj (.) 6 00E+05 4.00E+05 2 00E+05 O .OOE+OO 08 12 1 6 20 24 04 08 12 T i me of Day 48

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Fig. 6 PCC 7002 mRNA, RuBP and Carbon Fixation 30 0 5 0 4 5 25.0 4 0 :::; 3 5 20. 0 :::; c;, E :I 3 0 c;, .., 0 c: ::!:. 2 5 ... 15 0 0 "C ::!:. Cl) )( u:: 2 0 z ... 0: 0 10 0 E 0 1. 5 5 0 1. 0 0 5 0 0 0.0 08 12 16 20 24 04 08 12 16 20 Time of Day 49

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Fig. 7 Pavlova Growth Curve 1.80E+05 1 60E+05 1.40E+05 1 20E+05 ...J E 1 00E+05 Q) 8 00E+04 (.) 6 00E+04 4 00E+04 2 00E+04 O .OOE+OO 08 12 16 20 24 04 08 12 16 20 Time of Day 5 0

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Fig 8 Pavlova mRNA, RuBP and Carbon Fixation 9 .E+02 1 E-08 8 .E+02 9 .E-09 C02 fixation (tJJIL) B.E-09 Gl c: 7.E+02 -omRNA (ng/ml) (J 0 : ; A RubisCO Ereyme Activity/Cell 7 .E-09 "' 6 .E+02 )( : u u:: : 6 .E-09 N 5 .E+02 Gl 0 S .E-09 E () ..... >. '0 4 .E+02 N c: 4 .E-09 c: "' w 3 .E+02 3 .E-09 0 z () a:: 2 .E+02 VI E 2 .E-09 :0 :I 1 .E+02 I 1 .E-09 a:: O .E+OO O .E+OO 08 12 16 20 24 04 08 12 16 20 Time o f Day 51

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Fig. 9a Seward Johnson Lagrangian-Surface Drifters 5 0 .,--------------------------. 1 6 :J 4 5 4.0 0, 3 5 .s <( 3 0 z 2 5 g 2 0 IV 1 5 1.0 0 5 -+---Chromo mRNA -o-Cyano mRNA )i / \ / \ \ \ R I \ I \ I \ I \ I \ o.o 1533 0831 2121 1033 2022 1024 2208 TimeofDay 52 1.4 l 1 2 :J 0, c: 1 0 :; 0 8 / t 0 6 j 0.4 (..) 0 2 0 0 0758

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Fig. 9b Seward Johnson Lagrangian 15m Drifters ,----=----;===============;-------------,-4 0 0 -D Cyano mRNA -+-Chromo mRNA 0 ' ' p / t :: 1 2 5 2 0 0 1 5 E e 1 0 (..) 0 5 . : .____;_ o o 1015 2000 1020 2100 1030 2130 1000 Time of Day 53

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Fig. 10 Seward Johnson Vertical Profile 0 5 10 -E 15 .J:: / -m RNA Cyano a. 20 / Q) ./ *mANA Chromo 0 25 I / 30 ... 35 0 10 20 30 40 50 mANA (ng/L) 54

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Fig. 11 Pelican Lagrangian-mRNA and Carbon Fixation 200 0.6 0 180 -o--Cyano mRNA c ns -+-Chromo mRNA 0.5 >. 160 (.) --+-14C F ix ation ..r:. '-140 ....I 0-.J 0.4 --O"l _J C) 120 c C)-s:: C<{ 100 0.3 0 "l""Z co ciO:: 80 X -e u:: 0 0.2 N E 60 0 0 u '-.c 40 0 1 (.) 20 0 0 06 10 14 18 22 02 06 10 14 18 22 Time of Day 55

PAGE 64

800 a.: 700 0 ::s u. 600 "C Q) ...J 0:: E 500 "C r:: Q) C'IS c. 400 Q) en C')= r:: Q) EU 300 0 () 200 0 Ol 100 000 Fig. 12 Pelican Lagrangian-Autofluorescent Cell Counts r---1------------------0 .12 I \ I I I \ \ 06 10 14 --4--10) 6 -Orange Ruor Cells {x 100) Fluor. Cells (x 1 0) __._Chi a (ug/L) I I I A._. I .;r: ,.. ....... r I I I \ I I 18 22 02 06 10 14 Time of Day 56 \ 18 i 0 1 1 I J C') ::s ...... 1 0 .10-; >. .r:: c. 0.09 0 0 .08 0 .07 22 0 .r:: ()

PAGE 65

Fig. 13a Pelican Vertical Profile St 2-mRNA, DNA and Carbon Fixation 1 5 30 45 Depth (m) 58 70 95 0 mRNA and DNA (ng/L) 2 4 6 8 1 0 1 2 8 Cyan o mRN A ... Cyano DNA 14 C F ixation 110 lk>----4---1-----f-------l 0 0.5 1 1 5 2 C02 Fixation (ug/L/h) 57

PAGE 66

Fig 13b Pelican Vertical Profile St 2 -Autofluorescent Cell Counts 1 5 30 45 Depth (m) 58 70 95 0 DAPI BDC /ml 2 / / '\ I \ 3 4 5 6 DAPI BDC (105 ml-1) B Chl a ..t. Orange Fluor. C ells -)( Red Fluor. Cells 110 -+-1 -t-1 lt-----l 0 2 4 6 8 10 12 14 1 6 1 8 Orange and Red Cells/ml 58

PAGE 67

Fig. 14a Pelican Vertical Profile St 2 Cyano Clade Pigments Depth (m) Chlorophylls (ug/L) 0.0 0.1 0 2 0.3 0.4 0 .5 0 6 45 58 70 95 -1--t -1--0 02 0 .04 0 .06 0.08 0 10 0 1 2 0.14 Carotenoids (ug/L) 59

PAGE 68

Fi g 14b Pelican Vertical Profile St 2 Chromo Clade Pigm e nts Depth (m) Chlorophylls (ug/L) 0 00 0.03 0 05 0.08 0.10 0.13 15 30 4 5 58 70 95 t Chlorophylls c1 &c2 B Fucoxanthin -* '-Hexanoyloxyfucoxanth i n 1 9'-Butanoyloxyfucoxanthin .. : ... 0 00 0 03 0.06 0 09 0 12 0 15 0 18 Carotenoids (ug/L) 60

PAGE 69

Fig. 15a Pelican Vertical Profile St 3 Cyano and Diatom DNA and C02 Fixation Depth (m) 0 20 40 55 70 85 100 DNA (ng/L) 2 3 4 5 6 -lrChromo DNA 14C Fixation 115 -a:-.o--+ --+---t-----t------1 0 0 1 0 2 0 3 0.4 0 5 C02 Fixation (ug/Lih) 6 1

PAGE 70

Fig. 15b Pelican Vertical Profile St 3-Autofluorescent Cell Counts Depth (m) BDC/ml and Chi a (ug/L) 2 3 4 5 -+-DAPI BD C -Chi a 20 -:&-Orange Fluor Cells --*-Red Fluor Cells 40 55 70 85 100 115 0 0 2 5 5 0 7 5 10 0 12 5 1 5 0 Orange and Red Fluor. Cells/ml 62

PAGE 71

Fig. 16a Pelican Vertical Profile St 3 Cyano Clade Pigments Depth (m) Chlorophylls (ug/L) 0 0 .0 3 0 06 0 09 0 12 0 15 0 18 0 .21 0 24 0 27 --chlorop hyll a2 20 chlorophyll b2 lk Zeaxanthin 40 70 85 100 . . , ;o 115 0 0 02 0 04 0 .06 Carotenoids (ug/L) 63 0 08

PAGE 72

Fig. 16b Pelican Vertical Profile St 3-Chromo Clade Pigments Chlorophylls (ug/L) 0 .00 0 02 0 .04 0 .06 0 .08 20 + Chlorophylls c1 &c2 Fucoxanlh in A 19'-Hexanoyloxyfucoxanthin 40 19'-Butanoyloxyfucoxanthin 55 Depth (m) 70 85 100 115 0 .01 0.02 0 05 0 .08 0 .11 0 14 Carotenoids (ug/L) 64


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