carbon cycling: molecular regulation of photosynthetic carbon fixation

Microb Ecol (1996) 32:231-245 MICROBIAL ECOLOGY

© 1996 Springer-Verlag New York Inc.

Carbon Cycling: Molecular Regulation of Photosynthetic Carbon Fixation

J.H. Paul

kJepartrnent of Marine Science, University of South Florida, St. Petersburg, FL 33701, USA

Received: 22 November 1995; Accepted: 6 December 1995

Abstract, Photosynthetic carbon fixation by phytoplankton is a key component of the global carbon cycle. Our understanding of the types of picoplankton and ultraphytoplankton involved in this process is evolving. However, mechanisms of regulation of photosynthetic carbon fixation in the oceans are poorly understood. All phytoplankton fix CO2 by reductive carboxylation employing the enzyme ribulose bisphosphate carboxylase (RuBPCase). The sequence of the gene encoding the large subunit of the enzyme (rbcL) has been relatively conserved, with two major evolutionary groups among oxygenic photoautro- trophs: the cyanobacteria/green algae/higher plants and the chromophytic algae. Gene probes made from representative members of these groups have been used to study the transcriptional regulation of RuBPCase in natural phytoplankton populations. Levels of rbcL mRNA correlated with rates of photosynthetic carbon fixation. A diel pattern in both carbon fixation and levels of rbcL mRNA was observed, with greatest values for both during daylight hours. This data supports transcriptional regulation as a major mechanism for regulation of carbon fixation in the oceans. This approach can be used to measure expression of conserved genes encoding other important geochemical functions.

Introduction

Concern over global warming has caused renewed interest in the carbon cycle, particularly with respect to the oceans [75]. As the primary biological mechanism for CO2 uptake in the oceans, the role of phytoplankton in global warming has been a subject of debate and experimentation [54, 97]. The concentration of carbon dioxide in surface waters and the flux across the atmosphere-ocean interface is controlled by the photosynthetic activities of marine phytoplankton. Therefore, marine phytoplankton are the major controlling agents in the biogeochemistry of inorganic carbon in the surface waters of the world's oceans [71].

Phytoplankton utilize CO2 through photosynthetic carbon fixation (PCF), the process by which plant cells use light energy for reductive carboxylation that results in the primary formation of plant material (primary production). The carbon fixation pathway based on ribulose-l,5-carboxylase (RuBPCase) is a very old one, having

232 J.H. Paul

been in operation for at least 3.5 billion years [77]. In terms of understanding oceanic trophodynamics, the importance of this process cannot be overstated. The development of methodology to accurately measure this process has provided an unending challenge to ocean researchers [13, 26, 82].

The capability to study the regulation of PCF in phytoplankton populations with molecular biological techniques is currently evolving [48, 63, 64, 67]. The purpose of this review is to give an overview of this area, relating our own work to that of others.

Phytoplankton and the Importance of RuBPCase

The types of organisms responsible for autotrophic CO2-fixation in the world's oceans include cyanobacterial picoplankton [45, 52, 96], eucaryotic ultraphytoplankton [78, 79], prochlorophyte-like organisms [15, 16, 62, 94], and larger chromophytic phytoplankton (chain-forming diatoms, dinoflagellates, etc.). Our first understanding of autotrophic picoplankton was that these were primarily if not solely Synechococcus [43, 45, 52, 85, 96]. The discovery of prochlorophyte-like microoganisms [15, 16] suggested that these organisms might be the dominant primary producers in the water column. With the common use of flow cytometry in the study of water column microbial populations, it has been shown that the relative proportion of these forms in the water column is dependent upon geographic location, time of year, and depth in the water column. For example, Synechococcus- like picocyanobacteria have been shown to be dominant autotrophs in the Sargasso Sea [35], with prochlorophytes and ultraeucaryotes constituting a smaller proportion. Off Hawaii, prochlorophytes have been shown to be the dominant phototrophs [10]. In fact, of the bacterial (DAPI-positive) direct counts, 55% were shown to be autotrophic picoplankton, which questions the validity of the bacterial direct count technique in enumerating only heterotrophic bacterioplankton. Campbell et al. [10] have concluded that the relative importance of Prochlorococcus differs among ocean regions and varies inversely with Synechococcus abundance. In the Mediterranean, much of the photosynthetic carbon fixation was attributed to eucaryotic ultraphytoplankton [53]. Synechococcus was abundant in surface waters, the prochlorophytes had a subsurface maximum typically between 75 and 110 m, and the ultraphytoplankton had a constant abundance over the first 100 m. Courties et al. [19] found a green prasinophyte-like alga to be the dominant autotroph in a lagoon in the Mediterranean. This alga, named Ostreococcus tauri reached concentrations of 104 to 105 cells/ml. Others have found microeucaryotes to have an increasingly important role in primary production in coastal [22, 81, 87] and oceanic environments [66].

All pico- and phytoplankton are believed to fix CO2 primarily through C3 intermediates (e.g., 3-phosphoglyceric acid) by the action of RuBPCase [4, 18]. A small portion of carbon fixation occurs via [3-carboxylation (through the action of PEP carboxylase and PEP carboxykinase) in most microalgae and cyanobacteria. This is not a true C4 pathway because the carbon fixed into C4 products does not reappear in C3 intermediates [18].

RuBPCase is the pivotal enzyme in the pathway of carbon fixation of virtually all primary producers and may play a role in the modulation of CO2 levels on a

Regulation of Carbon Fixation 233

global scale. Current thinking indicates that CO2 is seldom if ever limiting for PCF in the world's oceans [70]. RuBPCase has a very slow specific reaction rate (<50 tool COJmol enzyme s -~) that is lower than any other enzyme of C assimilation, and lower than most enzymes in general. This means that even at saturating CO2 concentrations, a large fraction of cell protein must be devoted to RuBPCase to support the flow of carbon through metabolic pathways [70]. This is further complicated by the low affinity of the enzyme for CO2 and the competing oxygenase activity (see below).

In eucaryotes and virtually all procaryotes, RuBPCase is a large hexadecameric protein comprising equal numbers of small and large catalytic subunits (L8S8). The only known exception to this structure is the type II RuBPCase isolated from purple nonsulfur photosynthetic bacteria such as Rhodospirilum rubrum and Rhodobacter sphaeroides, which is composed of multimers of large subunits only (Lx) [30, 88, 90]. The large subunit is the major catalytic portion of the molecule, and some activity occurs with purified LS only [89]. The small subunit is not involved in substrate recognition, but can substantially affect key catalytic factors of the holoenzyme [89].

While the nucleotide sequence of the small subunit is divergent among photosynthetic organisms, the large subunit of type I (LsS,) RuBPCase is relatively conserved, with 80% and 70% similarity at the amino acid and nucleotide levels, respectively, for cyanobacteria to higher plants [80]. Chromophytic algae (chrysophytes and diatoms) [42, 72], rhodophytes [93], and Cryptomonas • [21] have been shown to contain divergent rbcL genes. In what has been termed the chlorophytic plants (containing chlorophyll a and b) [58], the rbcL gene is encoded on the chloroplast genome (ctDNA), and the rbcS (small subunit gene) is found in the nuclear genome. In chromophytes, rhodophytes, and Cryptomonas ci4, rbcL and rbcS are both encoded by the chloroplast genome [21, 41, 42, 72, 93], and in procaryotes the rbcL and rbcS genes are separated by a small noncoding region on the chromosomal genome [31, 88].

Regulation of Photosynthetic Carbon Fixation

The regulation of photosynthetic efficiency by pigment adaptation in response to long-term variation in light intensity and quality, termed photoacclimation [24, 48, 73], has been well studied both in ambient populations [29, 32, 35, 68, 94, 95] and laboratory cultures [3, 8, 46, 98]. These studies have shown that both cyanobacterial and prochlorophyte picoplankton are well acclimated for use of light energy throughout the photic zone [34, 68]. However, the mechanisms of short-term adaptation to rapidly changing.light fields, as might occur in a well-mixed water column, have not been determined.

The control of RuBPCase expression and activity is regulated by a myriad of mechanisms that vary among photosynthetic organisms, some of which are opera- tional only in higher plants, and others in all oxygenic photautotrophs examined. These include: (1) activation of the enzyme by CO2, which is the carbamylation of lysine 191; (2) enzyme activation and inhibition by phosphorylated metabolites of the Calvin cycle, most notably RuDP; (3) inhibition of RuBPCase by 2-carboxy arabinitol-l-phosphate (CAMP) in higher plants, a mechanism to limit RuBPCase

234 J.H. Paul

activity in the dark; (4) activation of the enzyme by RuBPCase activase, an enzyme that destabilizes the tight inhibitory binding of RuDP to RuBPCase; (5) posttranslational modification (both procaryotes and eucaryotes); (6) specific proteolytic diges- tion of RuBPCase (procaryotes and eucaryotes); and (7) control of transcription of both rbcS and rbcL mRNA (for reviews, see [36, 88]). The remainder of this review will focus on transcriptional regulation of RuBPCase.

Transcriptional regulation of RuBPCase and other photosynthetic genes (i.e., psbA) [9] in response to light has been known to occur in many photosynthetic organisms. In cyanobacteria, where the rbcL and rbcS genes are separated by an intervening sequence (93 bp for Anacystis 6301 and 545 bp for Anabeana 7120) [59, 89] cotranscription occurs. Further along the operon is the RuBPCase activase, which is apparently transcribed as a separate entity [89].

In cyanobacteria, the rbcL-rbcS operon is thought to be expressed constitutively [14]. However, evidence suggests strong diel regulation of transcription of several genes. For example, the nitrogenase activity of Synechococcus RF-1 was shown to be transcriptionally regulated, with nitrogenase genes being derepressed during dark periods [39, 40]. This work was also one of the first demonstrations of entrained or circadian rhythms in procaryotes. Further work on this organism showed that the nitrogenase and rbcL transcription were occurring oppositely, with rbcL transcription occurring in the light, and nif transcription in the dark [17]. Opposing nitrogenase and photosynthetic (actually oxygen evolution) activity has been shown to occur in response to light-dark cycles in the marine cyanobacterium Synechococcus Miami BG 043511 [47].

In eucaryotic microalgae, transcriptional regulation has also been demonstrated as a RuBPCase regulatory mode. For example, Steinbi[3 and Zetsche [84] found that light stimulated synthesis of rbcL transcripts in the green alga Chlorogonium. As well as transcriptional regulation, there is evidence of posttranscriptional regulation in Chlamydomonas reinhardtii [56] and Euglena gracilis [55]. Light sensitive regulatory sequences found upstream of the rbcS (nuclear-encoded) gene were not found in the intergenic regions of the rbcL-rbcS of Cylindrotheca. Thus, regulatory mechanisms are most likely different between chlorophytic phytoplankton and chromophytic phytoplankton.

Virtually nothing is known concerning the regulation of this enzyme in natural phytoplankton populations. Rivkin [74] has suggested that RuBPCase enzyme activity levels might be a useful indicator of photoadaptation in natural phytoplankton populations. The molecular regulation (i.e., short-term control) of this enzyme may be a key factor controlling production in the world's oceans and continental shelves. When studies of the absolute abundance of electron transport components and abundance of RuBPCase in algal cells in lab studies were performed, it was found that carbon fixation, not electron transport, was rate limiting in photosynthesis [86]. Variations in electron transport activity, including those of reaction centers, were not coupled to variations in carbon fixation capacity under conditions of nutrient and light saturation [25].

The molecular basis for die1 variations in photosynthetic efficiency (P~x; txg C fixed/tzg Chla) [23, 28] in natural phytoplankton populations is unknown. Diel variations in photosynthetic capabilities of natural phytoplankton communities have been known some time [12, 20, 69]. For example, Carpenter and Campbell [12] showed strong evidence for diel patterns in cell division in oceanic Synechococcus


populations. An approach to understanding the short-term regulation of diel photosynthetic processes is to look at transcriptional regulation of target genes with mRNA analysis.

Detection of Gene Expression by mRNA Analysis in Natural Phytoplankton Populations

The application of nucleic acid technology to microbial ecology has revolutionized our understanding of what organisms are present in aquatic and terrestrial ecosystems. These techniques have the potential to identify microbial components without the need to isolate or cultivate individual organisms [2, 37, 57]. Such procedures generally involve extraction of DNA from the community genome and direct probing with target sequences [33, 38, 44, 61], probing with another community's DNA [49-51], or amplifying distinct target sequences [5, 6, 83, 99].

Although these approaches give information on the diversity of organisms present, no information is given on the activity of these organisms. Beyond detection of a genotype in an ecosystem, it is essential to determine the activity of specific genotypes [11]. The detection of mRNA of target genes of interest has been successfully used to study expression of mercury resistance genes in soils [91, 92], rhythmic nitrogen fixation in Synechococcus RF-1 [39, 40], chloramphenicol acethltransferase expression in B. subtilis [1], expression ofpsbA genes in Synecho- coccus [9, 76], and hydrogenase gene expression in Alcaligenes eutrophus [60].

Advantages of detection of gene expression by mRNA analysis is that mRNA is very short lived, particularly in procaryotic cells (half lives as short as 2-5 min have been reported [7]). Thus, detection of message will ensure that the gene, and therefore the microorganisms from whence it came, has been recently active. A second advantage is that one basic technology can be employed to detect gene expression for many different conserved target genes. Once total planktonic RNA has been extracted and blotted, the blots can be washed and reprobed with other probes for other environmentally significant genes.

We have developed a method for the extraction of microbial mRNA from seawater [64, 65, 67]. The system is based upon guanidinium isothiocyanate extraction of mRNA from filtered samples, followed by blotting and probing with single strand RNA probes [27] (Fig. 1). RNA probes are generated by cloning the target gene of interest into Riboprobe vectors that contain unique RNA polymerase promoters (Sp6 and T7) flanking the gene of interest. We have subcloned the rbcL genes from Synechococcus PCC6301 and Cylindrotheca sp. strain N1 into such vectors. The Synechococcus probe should detect all cyanobacteria, green algae, and prochlorophytes, whereas the diatom probe should detect all diatoms, cryptomonads, red algae, and dinoflagellates.

Figure 2 shows the use of such probes to detect rbcL gene expression in phytoplankton populations from Bimini Harbor and a mangrove-fringed lagoon east of Bimini Harbor. All probing was under stringent conditions (in 50% formamide at 55°C for RNA targets and 42°C for DNA targets [65]). The Synechococcus probe hybridized strongly to the RNA from both samples, whereas the diatom probe hybridized primarily with the sample from the mangrove-fringed area. We interpret this data to indicate that the mangrove-fringed area contained chromophytic algae

236 J.H. Paul

DNA Isolation

1. Add 1%SDS-STE buffer

2. Boil 2 min.

3. Cfg. and collect supernatant

Precipitate

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2. Add phenol, mix

3. Cfg. and collect upper phase

Precipitate Resuspend and separate into aliquots

for enzyme digest controls

DNA 1 l RNA Dot Blot

DNA ] RNA

Hybridize W/ single-stranded RNA probes

DNA RNA

Quantitate radioactivity Fig. 1. Outline of the "gene expression per gene dose" protocol for measurement of gene expression in naturally occurring picoplankton populations. This methodlology has been used to measure expression of the rbcL gene in phytoplankton populations.

as well as chlorophytic, while the Bimini Harbor sample contained mostly chlorophytic-type organisms. This agrees with microscopic observations of shallow mangrove environments, where diatoms and dinoflagellates are more prevalent than picocyanobacteria, which dominate open bays (Paul et al., unpublished observations).

We have combined our RNA extraction procedure with a DNA extraction protocol to determine a specific measure of gene expression: gene expression per gene dose (Fig. 1). This gives a measure of the target gene population. In studies with pure cultures, gene expression is normalize to rRNA content. However, this is not

Regulation of Carbon Fixation

Cyano rbcL Diatom rbcL

237

A B A B

U

D AS

R

U

D S

R

Fig. 2. Dot blot of mRNA extracted from natural phytoplankton populations in the center of Bimini Harbor, Bahamas (columns labeled A) and a shallow mangrove-fringed bay east of Bimini (columns labeled B), probed with the Synechococcus probes (Cyano rbcL) or the Cylindrotheca sp. (Diatom rbcL) probes. Duplicate samples were taken for each treatment. The samples were probed with both the antisense (group of three rows labeled AS) and sense (group of three rows labeled S) RNA probes. Rows labeled U are undigested, whereas those labeled D and R are DNAse and RNase digested samples, respectively.

possible with natural populations, where the rRNA signal would come from many nontarget organisms. For example, increases in a target mRNA may be caused by increased transcriptional activity (and thus gene expression) or increases in the target population size. Dividing mRNA abundance by target DNA concentration normalizes gene expression for target population size. We have detected rbcL gene expression in natural phytoplankton of the Southeast Gulf of Mexico (Fig. 3). Analysis of samples taken from along transect from the estuarine environment of Tampa Bay to the offshore environment indicated that gene expression followed carbon fixation and phytoplankton abundance, indicating that we could efficiently detect gene expression in waters of a variety of trophic states (Fig. 4).

We have also performed these analyses in a profile at an oligotrophic station in the Gulf of Mexico. Figure 5 shows the results of the data plotted as a function of depth. At this station, cyanobacterial picoplankton direct counts decreased as a function of depth, while red fluorescing cells (presumably eucaryotic ultraphytoplankton) increased with depth (Fig. 5a). Also plotted is the rate of carbon fixation at a fixed light intensity and chlorophyll a concentration (Fig. 5b). The rbcL mRNA had a subsurface maximum and then decreased with depth, while rbcL DNA decreased rapidly with depth from a surface maximum (Fig. 5C). The specific level of rbcL gene expression, rbcL mRNA per rbcL DNA, showed a maximum

238 J.H. Paul

4

Gulf of Mexico

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28°N

27

26 Fig. 3. Locations for stations sampled for rbcL mRNA in the southeastern Gulf of Mexico. Station 3 was the sampling location for the diel study, whereas station 6 was the location for sampling in vertical profile.

at 60 m, which was reflected in specific rates of carbon fixation (i.e., txg C fixed/ Ixg Chl a, Fig. 5d).

Figure 6 shows the results of a diel study performed by enclosing 150 liters of oligotrophic Gulf of Mexico surface water in a ship deck surface incubator. The water was maintained at in situ temperature (27.5°C) with a chiller system. Again, Chl a, rbcL mRNA, rbcL DNA, 14C-carbon fixation, and specific levels of carbon fixation (~g C fix/txg Chl a) and rbcL expression (ng mRNA/ng DNA) were measured. A strong diel pattern in specific levels of rbcL gene expression and carbon fixation were noted, with maxima at noon and minima at midnight (Fig. 6). These results suggest that diel variations in in situ carbon fixation capacity are regulated at the level of transcription of the RuBPCase genes.

Summary and Future Prospects

Our studies on the regulation of RuBPCase in natural phytoplankton populations indicate that control is at the level of transcription. Other mechanisms of regulation may be occurring as well. Sensitive methods for detection of RuBPCase protein levels are needed to determine how enzyme levels change over time and to detect posttranslational regulation. The data presented here provide only preliminary evidence for regulation of this enzyme in natural populations.

The regulation of geochemically important, microbially-mediated processes at the cellular level is a new field in the growing discipline of molecular ecology. The example given above is a first attempt to apply this approach to one aspect of the carbon cycle. The rbcL gene lends itself to this type of study because of


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20 ~ and rbcL mRNA (Synechoeoccus probe) and photosynthetic carbon fixation (B) at the stations indicated in

l 0 ~ Fig. 3. Pico cells refers to orange-fluorescing picocyanobacteria, and red

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its conserved nature, and other genes may not be as ideally suited to this approach. We have extended these studies to include amplification, cloning, and sequencing of the transcriptionally active rbcL genes in the water column [66]. This research will enable identification of the active players in carbon fixation in the water column.

We have focused this review on transcriptional regulation of rbcL, yet this is only one of five or six regulatory mechanisms for RuBPCase activity, and how transcriptional regulation fits with the other mechanisms in natural populations is completely unknown. Additionally, the photosynthetic machinery of cells (photosynthetic reaction centers, electron transport carriers, primary and accessory pig-

240 J.H. Paul

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ments) are also under some type of regulatory mechanism(s). How these processes are regulated are fertile areas of research for future studies.

Another cellular process that may be important to the global carbon cycle is the CO2 concentrating mechanism (CCM) of phytoplankton cells [70]. As pointed out, RuBPCase is an extremely inefficient enzyme, even at saturating levels of CO2. Because CO2 concentrations in seawater are well below the Km for RuBPCase, nearly all phytoplankton cells have developed mechanisms to concentrate CO2 at


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Fig. 6, Diel variations in rbcL mRNA and related parameters for phytoplankton collected in a 150- liter sample from station 3 and incubated in a deck-top incubator. A Photosynthetic carbon fixation and rbcL mRNA (Synechococcus probe), B chlorophyll a and rbcL DNA, C specific rates of carbon fixation and rbcL gene expression per gene dose.

the intracellular site of RuBPCase activity. These CCMs are also regulated in response to ambient HCO3- are dissolved CO2 concentrations [70]. Thus, the CCMs may have a more direct link with the global carbon cycle than RuBPCase. However, how the CCMs respond to the cellular demand for CO2 put on them by RuBPCase is also not known.

The study of biogeochemical processes by molecular techniques is a fruitful area for investigation of marine and terrestrial environments. Through such novel and multidisciplinary studies we will improve our understanding of our changing planet.

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