Physiology

Morning Session 1: Physiology (0900-10:30)
Chair: Bethany Jenkins

0900-0930 (Invited Speaker)
THE PHYSIOLOGY AND MOLECULAR BIOLOGY OF DIATOM CO2 CONCENTRATING MECHANISMS
Brian M. Hopkinson1, Chris. L. Dupont2, C. Shen1, and Y. Matsuda3
1Department of Marine Sciences, University of Georgia, Athens, GA, USA
2J. Craig Venter Institute, San Diego, CA, USA
3Department of Bioscience, Kwansei-Gakuin University, Gakuen, Sanda, Hyogo, Japan

Like most microalgae, diatoms operate a CO2 concentrating mechanism (CCM), which elevates the concentration of CO2 at the site of fixation, to overcome inefficiencies of the CO2 fixing enzyme RubisCO. The CCM is necessary to maintain high rates of photosynthesis at low CO2 concentrations, such as those found in the ocean and many other environments where diatoms thrive. However, it is down-regulated at high CO2 and plays a key role in the response of diatoms to ocean acidification. With the development of new molecular and physiological approaches, we have made surprisingly rapid advances in our understanding of the CCM in marine diatoms, most notably in the model diatom Phaeodactylum tricornutum. In P. tricornutum, the core components of the CCM have been identified, including bicarbonate transporters and carbonic anhydrases, and an integrative model of the system has been developed. High rates of bicarbonate transport into the chloroplast drive the major CO2 gradients in P. tricornutum, elevating CO2 around RubisCO and creating a CO2 deficit in the cytoplasm that drives uptake from the environment.  In other diatoms the CCM is less well characterized. While some CCM components appear to be conserved among diatoms, other features differ suggesting the CCM may work in fundamentally different ways in some species.

0930-0950
THE COST TO OPERATE CARBON CONCENTRATING MECHANISMS IN DIATOMS RANGES DRAMATICALLY ACROSS SPECIES AND TEMPERATURES DUE TO WIDE VARIABILITY IN RUBISCO KINETICS.
Jodi Young1, Brian Hopkinson2, Ana Heureux3, Robert Sharwood4, Spencer Whitney4, Francois Morel1
1 Geosciences, Princeton University, Princeton, NJ, USA
2 Department of Marine Sciences, University of Georgia, Athens, GA, USA
3 University of Oxford, Oxford, United Kingdom
4 Australian National University, Canberra, Australia

All diatoms possess a carbon concentrating mechanism (CCM) that saturates CO2 around the carboxylating enzyme, Rubisco. Without a CCM, diatoms would be CO2‐limited due to concentrations in the surface ocean often being much lower than Rubisco’s CO2 half saturation constant (KmCO2). The high-energy demand to operate the CCM has resulted in speculation that increasing anthropogenic CO2, and subsequent down-regulation of the CCM, could lead to increased primary production, though experimental results thus far have been conflicting. This study demonstrates that the energetic cost of the CCM varies greatly between different diatom species and temperature, largely due to the wide variation of Rubisco KmCO2 (ranging from 12 – 65 μM) and may provide a mechanistic explanation for the conflicting experimental results observed. Variations in Rubisco KmCO2 dramatically alter the concentration of intracellular CO2 required to saturate Rubisco. Using a model of a diatom CCM, we were able to calculate the extent of CCM down-regulation that would be expected in response to rising CO2 in different species and across latitudes, and speculate under which conditions is the change dramatic enough to potentially result in an increase of production.

0950-1010
AQUAPORINS IN TWO MARINE DIATOMS, PHAEODACTYLUM TRICORNUTUM AND THALASSIOSIRA PSEUDONANA AS A CHANNEL FOR CO2 AND NH3
Hiroaki Matsui, Kensuke Nakajima and Yusuke Matsuda
Department of Bioscience, Kwansei-Gakuin University, Sanda, Hyogo 669-1337, Japan

Inorganic carbon acquisition system in eukaryotic algae is still largely unknown except that some plasmamembrane type bicarbonate transporter were identified in the green alga, Chlamydomonas reinhardtii and the marine diatom, Phaeodactylum tricornutum. Carbon dioxide has been thought to be a highly permeable molecule against biological membrane, but recently such hydrophobic molecule was also found to require a specific channel to pass across biological membrane under marginal concentration gradient. Aquaporin (AQP) is one of candidates for such channel, which is known in some organisms to pass CO2 and NH3. The genomes of P. tricornutum and T. pseudonana respectively have 5 and 2 homologs of AQP gene, which are similar to barley AQP, HvPIP2;1. Quantification of transcripts by qPCR indicated that two AQP homologs PtAQP2 and TpAQP2 were increased in high CO2 and NH3, strongly suggesting that the function of diatom AQPs are related to CO2 and NH3. These AQP cDNAs were fused with the enhanced green fluorescence gene, egfp and were expressed in P. tricornutum or T. pseudonana cells. PtAQP2 and TpAQP2 were respectively localized at the plasmalemma, and the chloroplastic envelopes, suggesting that these AQPs are involved in acquisition of CO­2 and NH3 towards the chloroplast.

1010-1030
ENERGETIC COUPLING BETWEEN PLASTIDS AND MITOCHONDRIA DRIVES CO2 ASSIMILATION IN DIATOMS
B. Bailleul1,2,3,4, N. Berne1, O. Murik4, D. Petroutsos5, J. Prihoda4, A. Tanaka4, V. Villanova6, R. Bligny5, S. Flori5, D. Falconet5, A. Krieger‐Liszkay7, S. Santabarbara8, F. Rappaport3, P. Joliot3, L. Tirichine4, P. G. Falkowski2, P. Cardol1, C. Bowler4, G. Finazzi5
1 Génétique et physiologie des microalgues, Université de Liège, B‐4000 Liège, Belgium.
2 Environmental Biophysics and Molecular Ecology Program, Rutgers University, New Brunswick, NJ 08901, USA.
3 Institut de Biologie Physico‐Chimique (IBPC), UMR 7141, UPMC/CNRS, 13 Rue Pierre et Marie Curie, F‐75005 Paris, France.
4 Ecology and Evolutionary Biology Section, IBENS/CNRS, UMR 8197, 46 Rue d'Ulm, F‐75005 Paris, France.
5 UMR 5168, CNRS/CEA/INRA, iRTSV, CEA Grenoble, F‐38054 Grenoble Cedex 9, France.
6 Fermentalg SA, F‐33500 Libourne, France.
7 Institute for Integrative Biology of the Cell (I2BC), CEA/CNRS, Université Paris‐Sud, F‐91191 Gif‐sur‐Yvette cedex, France.
8 Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via Celoria 26, I‐20133 Milan, Italy.

Today the proportion of planetary primary production performed by diatoms is equivalent to that of terrestrial rainforests but the modes of regulation of the photosynthetic process in diatoms is still poorly known. In photosynthesis, the efficient conversion of CO2 into organic matter requires a tight control of the ATP/NADPH ratio which, in other photosynthetic organisms, relies principally on a range of plastid‐localized ATP generating processes. Using RNAi mutants and new biophysics tools, we show that diatoms regulate ATP/NADPH through extensive energetic exchanges between plastids and mitochondria. First, a significant proportion of the reducing power generated in the plastid is rerouted towards mitochondria. Second, some of the ATP generated in the mitochondria is imported in the plastid, which is efficient enough to generate a proton gradient across the thylakoid in the dark. Those energetic exchanges are mandatory for optimized carbon fixation and growth. We propose that this process, elucidated in Phaeodactylum tricornutum as well as in 4 other diatoms, may have contributed to the ecological success of diatoms in the ocean.

BREAK: 1030-1100

Morning Session 2: Physiology (1100-1230)
Chair: Angela Falciatore

1100-1230 (Invited Speaker)
DIATOMS IN A CHANGING WORLD
Zoe V. Finkel
Canada Research Chair in Marine Environmental Ecology, Mount Allison University, Sackville, New Brunswick, Canada, E4L 1A7

Climate change is expected to influence phytoplankton biomass and community composition and consequently impact food web structure and elemental cycling in the sea.  In particular, cell size and the elemental composition of phytoplankton cells within communities are sensitive to changes in environmental conditions. I will discuss some of the recent evidence for how diatom and other phytoplankton groups respond to selected environmental and climatic conditions over a range of physiological, ecological, and evolutionary timescales.

1130-1150
SIDEROPHORE-BASED IRON UPTAKE IN PHAEDOACTYLUM TRICORNUTUM
Chris Bowler, Javier Paz-Yepes, Emmanuel Lesuisse and Robert Sutak
Ecology and Evolutionary Biology Section, Institut de Biologie de l’Ecole Normale Supérieure (IBENS), Paris, France

Iron is essential for respiration and numerous other cellular functions. Phytoplankton must also maintain the iron-rich photosynthetic electron transport chain, which likely evolved in the iron-replete reducing environments of the Proterozoic ocean. The subsequent rise in oxygen since those times has drastically decreased the levels of bioavailable iron, likely selecting for efficient iron acquisition mechanisms. Mesoscale iron fertilization experiments result in blooms dominated by diatoms, indicating diatoms can adapt to iron-limited waters. Yet the genetic and molecular bases are unclear. To address this topic, we are studying the iron starvation induced proteins (ISIPs) in the marine diatom Phaeodactylum tricornutum. We found that the product of the most induced gene in response to iron limitation, ISIP1, is involved in iron uptake via a siderophore-mediated mechanism. We show that P. tricornutum is able to grow on hydroxamate siderophores, while knocking down ISIP1 decreases siderophore uptake, resulting in impaired growth on that iron source.  We identified strong siderophore localization near the chloroplast using a fluorescently-labelled artificial siderophore, which is significantly reduced in the ISIP1 knockdown line. ISIP1-YFP localizes to the cell surface and the chloroplast membrane, suggesting that ISIP1 might also be involved in chloroplast siderophore uptake. ISIP1 is expressed by diverse marine phytoplankton, indicating that it is an ecologically significant adaptation in the marine environment.

1150-1210
THE NOVEL PROTEIN LOCALIZED IN PYRENOID-PENETRATING THYLAKOID IN A MARINE DIATOM, PHAEODACTYLUM TRICORNUTUM
Sae Kikutani1, Ai Miyatake1, Chikako Nagasato2, Yusuke Matsuda1
1Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, Hyogo, 669-1337, Japan
2Muroran Marine Station, Field Science Center for Northern Biosphere, Hokkaido University, Muroran 051-0003, Japan

Pyrenoids are protein aggregates where most RubisCO is located and are thought to play a central role for CO2-concentrating mechanism (CCM) in eukaryotic algae. In the marine diatom Phaeodactylum tricornutum, it has been demonstrated that the pyrenoid contains RubisCO, two β-carbonic anhydrases, and two fructose-1,6-bisphosphate aldolases, but this is only a part of biochemical structure of the pyrenoids and its components and functions are still unclear. LciB is found as one of low-CO2 inducible genes in the green alga Chlamydomonas reinhardtii. LCIB/C complex was localized in the pyrenoid of C. reinhardtii and its vital function under moderate CO2 limitation was previously demonstrated. Four LCIB homologues were also found in diatom genome. LCIB homologues in diatoms may play a similar role as that in C. reinhardtii in the pyrenoid. In this study, we characterized these genes in P. tricornutum. One of the LCIB homologues, Pt43233, is localized in the pyrenoid-penetrating thylakoid. Down-regulation of pt43233 resulted in significantly decreased photosynthetic affinity for DIC in air-grown cells. These results suggest that Pt43233 plays a key role in CCM and pyrenoid-penetrating thylakoid in diatoms has a distinct function that is crucial for the CCM.

1210-1230
PHYTOCHROME-MEDIATED FAR RED LIGHT RESPONSES IN MARINE DIATOMS
Antonio Emidio Fortunato1, Marianne Jaubert1, Gen Enomoto2, Jean-Pierre Bouly1, Michael Thaler1, Raffaella Raniello3, Shruti Malviya4, Fabrice Rappaport5, Alessandra Carbone1, Chris Bowler4, Maurizio Ribera d’Alcalà3, Masahiko Ikeuchi2, Angela Falciatore1
1 Sorbonne Universités, UPMC Univ Paris 06, UMR 7238, Computational and Quantitative Biology, Paris, France.
2 Department of Life Sciences (Biology), Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan
3 Stazione Zoologica Anton Dohrn of Naples, IT
4 Institut de Biologie de l’Ecole Normale Supérieure (IBENS), UMR8197, Paris, France
5 Institut de Biologie Physico-Chimique, UMR 7141 CNRS-UPMC, Paris, France

Phytochromes represent a major class of red/far-red light photoreceptors widely diffused in nature and also described in several marine organisms. However, because of the strong red and far-red light attenuation along the water column, the relevance of marine phytochromes is still debated. Here, we explore the functional properties of phytochromes in diatoms. Biochemical analyses of Phaeodactylum tricornutum (PtDph) and Thalassiosira pseudonana diatom phytochromes (TpDph) provided the first evidence that Dphs are red/far-red light photoreceptors, acting as far-red light-activated protein kinases. Gene expression analyses in P. tricornutum revealed far-red light-mediated short-term responses that are abolished in transgenic lines with modulated PtDph content. An extensive phylogenetic analysis including several novel Dphs revealed the existence of diverse structural variants in centric and pennate diatoms, supporting widespread Dphs specialization in the marine environment. This, confronted to the scarcity of underwater red and far-red lights, prompted us reconsidering the underwater Phy utilizable radiation. Computational modelling indicated that far-red light from external and internal sources (i.e., chlorophyll autofluorescence) are both potential triggers of PtDph activity. Overall, our work provides new insights to revisit Dph activity and the role of far-red light signals in the marine context.