Resource utilization by phytoplankton: is nitrogen allocation amongst functional catalysts optimized in response to resource limitation?

Lead Research Organisation: University of Essex
Department Name: Biological Sciences


We have entered an era in biological oceanographic research when the information within the genomes of an increasing number of marine organisms is becoming increasingly available. One of the challenges facing biological oceanographers is to exploit this information to obtain greater insight into the functioning of marine ecosystems. Recently, Raleigh Hood and coworkers have posed the questions: o 'What role do all these new genes and proteins (identified by genomic approaches) play in driving marine ecosystem dynamics and biogeochemical cycles? o Which are important and which are not? o What role are they likely to play in the evolution of marine microbial communities, how might they have influenced global biogeochemical cycles over Earth's history, and how might they do so in the future.' (Oceanography, Vol 20, No 2 page 155) These are very challenging questions. We propose to take a small but important step in addressing a subset of issues raised by these questions. Our focus is on one representative of the marine phytoplankton, namely the marine coccolithophore Emiliania huxleyi. Emiliania is one of the thousands of phytoplankton species that contribute to photosynthesis in the sea. As a photosynthetic organism, she sits at the base of the food web that leads to fish and top predators including marine mammals and man. Emiliania is particularly useful to us in the genomic age of oceanographic research because she is one of the few phytoplankton species for which the entire genome is currently available. ( The genome sets the limits on the capability of an organism to exploit its environment. However, the genome represents an organism's potential rather than what is actually achieved in a given situation. How an organism exploits the environment becomes manifest in the composition of its proteome. The proteome consists of all of the proteins that are manufactured by a cell. The proteome is not a static entity. Rather, the proteome is a dynamic entity that is reorganized in response to changes in the environment. Of particular interest are changes in the proteome that increase the ability of an organism to obtain resources from the environment and use these resources for growth and reproduction. Also of importance, are changes in the proteome that protect an organism from environmental stress. Growth is promoted when resources are plentiful. These resources include light and inorganic nutrients. Growth is limited when these resources become scare, or when environmental conditions deteriorate. In particular, light is an important limiting factor on seasonal time scales (low-light in winter versus high-light in summer) and with increasing depth in the sea. Nitrogen is the main limiting factor for phytoplankton growth in over 50% of the surface of the sea in summer, with phosphorus an important secondary limiting factor in many regions. Advances in technology now allow both qualitative and quantitative measurements of how the proteome changes in response to environmental factors. Documenting changes in the proteome provides a way to assess how the state of a cell such as Emiliania changes. Our goal is to document changes in the abundance of proteins associated with different bioenergetic and biochemical pathways or functions. This will allow us to assess the cost of acclimation in terms of changes in the proportions of cell biomass amongst these pathways/functions. The goal of our research is to employ this new information to inform a cost-benefit analysis of acclimation within Emiliania huxleyi. Ultimately, this information will contribute to our understanding of adaptation of marine phytoplankton to the range of environmental conditions encountered in the sea.
Description Four experiments were undertaken, each involving an extended period of phytoplankton culturing to insure that cells were in balanced growth, followed by extensive analytical work and proteomics analyses, followed by data reduction.

Experiment I: Emiliania huxleyi was grown to steady-state in high (1000 ?mol photons m-2 s-1) and low (30 ?mol photons m-2 s-1) light. The major conclusion of this experiment was that clear differences in photophysiology were reflected by changes in the relative abundances of photosynthetic proteins. Most notable were changes in the abundances of different chlorophyll-fucoxanthin binding proteins associated with light harvesting and photoprotection. However, proteins associated with biosynthesis were largely unaffected by the light treatment.

Experiment II: Acclimation to N and P limitation. Emiliania huxleyi was grown under three treatments (nutrient replete, N-limited, P-limited). Changes in cellular elemental composition (C, N, P) biochemical composition (protein, RNA, lipids) were consistent with previous reports of responses to N and P limitation. The major conclusions are that (i) most components of the proteome were unaffected by nutrient limitation, however, (ii) cell surface proteins associated with N uptake increased under N limitation and those associated with P-uptake increased under P-limitation. Two manuscripts are in preparation. The first manuscript describes the response of the proteome as a whole, together with the biochemical and elemental analyses, focusing on the response of the cell surface proteins to nutrient limitation. A manuscript describing this experiment is expected to be submitted by December 2012 (Introduction, Methods, Results are complete, and there is an outline of the discussion). The second manuscript will focus on the photophysiological responses to nutrient limitation and the corresponding changes in the proteome.

Experiment III: Acclimation to pCO2 and inorganic N source (nitrate versus ammonium). This experiment was undertaken in collaboration with Dr Stephane Lefebvre at San Francisco State University. We took advantage of the opportunity to undertake the proteomic analysis of samples from 2 CO2 treatments X 2 nitrogen sources X 2 times of day to provide data for comparison with a transcriptomics analysis undertaken by Dr Lefebvre. To the best of our knowledge, this will be the first comparison of responses of both the transcriptome and proteome in a microalga, allowing an initial assessment of transcriptional versus translational controls of cell physiology.

Experiment IV: Acclimation to four light intensities. This experiment complements Experiment 1 by providing greater resolution of the response to light intensity, and in addition provided a sample set to improve the absolute quantitation of the abundances of specific proteins of known function. The main outcome of this experiment was to show that the response observed in Experiment I between very low and very high light intensities was associated with the effect of light intensity when cells were growing at their maximum growth rate.
Exploitation Route Academics are the main end users who will benefit from this research; the relevant disciplines range from algal physiology to ocean biogeochemistry and ecosystem and ecological modelling. The main benefit will be access to our research findings through peer-reviewed scientific papers, and through our data sets.

Importantly given the funding of research into Ocean Acidification by NERC, the EU and the US NSF amongst others, our data on response of E huxleyi proteome to CO2 and inorganic N source, undertaken through a collaboration with researchers at San Francisco State University, provides significant new data that can feed directly into the current efforts to evaluate the implications of ocean acidification on primary productivity and biogeochemical cycles. This will provide highly relevant information that can be used by government agencies and policy makers, ultimately feeding into governmental directives addressing climate change.

Another important group of end-users is ecosystem modellers who are increasingly using optimality principles in constructing representations of phytoplankton growth. Insights gained from this research have helped to inform optimality based models of phytoplankton growth that have been published recently by Talmy and co-workers including Geider.
Sectors Environment