Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

Several cosmopolitan marine picoeukaryotic phytoplankton are B1 auxotrophs requiring exogenous vitamin B1 or precursor to survive. From genomic evidence, representatives of picoeukaryotic phytoplankton (Ostreococcus and Micromonas spp.) were predicted to use known thiazole and pyrimidine B1 precursors to meet their B1 demands, however, recent culture-based experiments could not confirm this assumption. We hypothesized these phytoplankton strains could grow on precursors alone, but required a thiazole-related precursor other the well-known and extensively tested 4-methyl-5-thiazoleethanol. This hypothesis was tested using bioassays and co-cultures of picoeukaryotic phytoplankton and bacteria. We found that specific B1-synthesizing proteobacteria and phytoplankton are sources of a yet-to-be chemically identified thiazole-related precursor(s) that, along with pyrimidine B1 precursor 4-amino-5-hydroxymethyl-2-methylpyrimidine, can support growth of Ostreococcus spp. (also Micromonas spp.) without B1. We additionally found that the B1-synthesizing plankton do not require contact with picoeukaryotic phytoplankton cells to produce thiazole-related precursor(s). Experiments with wild-type and genetically engineered Ostreococcus lines revealed that the thiazole kinase, ThiM, is required for growth on precursors, and that thiazole-related precursor(s) accumulate to appreciable levels in the euphotic ocean. Overall, our results point to thiazole-related B1 precursors as important micronutrients promoting the survival of abundant phytoplankton influencing surface ocean production and biogeochemical cycling.

Although the decision to proceed through cell division depends largely on the metabolic status or the size of the cell, the timing of cell division is often set by internal clocks such as the circadian clock. Light is a major cue for circadian clock entrainment, and for photosynthetic organisms it is also the main source of energy supporting cell growth prior to cell division. Little is known about how light signals are integrated in the control of S phase entry. Here, we present an integrated study of light-dependent regulation of cell division in the marine green alga Ostreococcus. During early G1, the main genes of cell division were transcribed independently of the amount of light, and the timing of S phase did not occur prior to 6 hours after dawn. In contrast S phase commitment and the translation of a G1 A-type cyclin were dependent on the amount of light in a cAMP–dependent manner. CyclinA was shown to interact with the Retinoblastoma (Rb) protein during S phase. Down-regulating Rb bypassed the requirement for CyclinA and cAMP without altering the timing of S phase. Overexpression of CyclinA overrode the cAMP–dependent control of S phase entry and led to early cell division. Therefore, the Rb pathway appears to integrate light signals in the control of S phase entry in Ostreococcus, though differential transcriptional and posttranscriptional regulations of a G1 A-type cyclin. Furthermore, commitment to S phase depends on a cAMP pathway, which regulates the synthesis of CyclinA. We discuss the relative involvements of the metabolic and time/clock signals in the photoperiodic control of cell division.

In large regions of the open ocean, iron is a limiting resource for phytoplankton. The reduction of iron quota and the recycling of internal iron pools are among the diverse strategies that phytoplankton have evolved to allow them to grow under chronically low ambient iron levels. Phytoplankton species also have evolved strategies to cope with sporadic iron supply such as long-term storage of iron in ferritin. In the picophytoplanktonic species Ostreococcus we report evidence from observations both in the field and in laboratory cultures that ferritin and the main ironbinding proteins involved in photosynthesis and nitrate assimilation pathways show opposite diurnal expression patterns, with ferritin being maximally expressed during the night. Biochemical and physiological experiments using a ferritin knock-out line subsequently revealed that this protein plays a central role in the diel regulation of iron uptake and recycling and that this regulation of iron homeostasis is essential for cell survival under iron limitation.

These authors contributed equally. SUMMARYWith fewer than 8000 genes and a minimalist cellular organization, the green picoalga Ostreococcus tauri is one of the simplest photosynthetic eukaryotes. Ostreococcus tauri contains many plant-specific genes but exhibits a very low gene redundancy. The haploid genome is extremely dense with few repeated sequences and rare transposons. Thanks to the implementation of genetic transformation and vectors for inducible overexpression/knockdown this picoeukaryotic alga has emerged in recent years as a model organism for functional genomics analyses and systems biology. Here we report the development of an efficient gene targeting technique which we use to knock out the nitrate reductase and ferritin genes and to knock in a luciferase reporter in frame to the ferritin native protein. Furthermore, we show that the frequency of insertion by homologous recombination is greatly enhanced when the transgene is designed to replace an existing genomic insertion. We propose that a natural mechanism based on homologous recombination may operate to remove inserted DNA sequences from the genome.

The default fate for eggs from many species is death by apoptosis and thus, successful fertilization depends upon suppression of the maternal death program. Little is known about the molecular triggers which activate this process or how the fertilization signal suppresses the default maternal apoptotic pathway. The MAP kinase (MAPK) family member, ERK, plays a universal and critical role in several stages of oocyte meiotic maturation, and fertilization results in ERK inactivation. In somatic cells, ERK and other MAPK family members, p38 and JNK, provide opposing signals to regulate apoptosis, however, it is not known whether MAPKs play a regulatory role in egg apoptosis, nor whether suppression of apoptosis by fertilization is mediated by MAPK activity. Here we demonstrate that MAPKs are involved in starfish egg apoptosis and we investigate the relationship between the fertilization induced signaling pathway and MAPK activation. ERK is active in post-meiotic eggs just until apoptosis onset and then p38, JNK and a third kinase are activated, and remain active through execution. Sequential activation of ERK and p38 is necessary for apoptosis, and newly synthesized proteins are required both upstream of ERK and downstream of p38 for activation of the full apoptotic program. Fertilization causes a dramatic rise in intracellular Ca2+, and we report that Ca2+ provides a necessary and sufficient pro-survival signal. The Ca2+ pathway following fertilization of both young and aged eggs causes ERK to be rapidly inactivated, but fertilization cannot rescue aged eggs from death, indicating that ERK inactivation is not sufficient to suppress apoptosis.

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Downregulation of MAP kinase is a universal consequence of fertilization in the animal kingdom. Here we show that oocytes of the starfishes Astropecten aranciacus and Marthasterias glacialis complete meiotic maturation and form a pronucleus when treated with 1-methyladenine and then complete DNA replication and arrest at G2 if not fertilized. Release of G2 by fertilization or a variety of parthenogenetic treatments is associated with inactivation of MAP kinase. Prevention of MAP kinase inactivation by microinjection of Ste11-DeltaN, a constitutively active budding yeast MAP kinase kinase kinase, arrests fertilized eggs at G2 in either the first or the second mitotic cell cycle, in a dose-dependent manner. G1 arrest is never observed. Conversely, inactivation of MAP kinase by microinjection of the MAP kinase-specific phosphatase Pyst-1 releases mature starfish oocytes from G2 arrest. The role of MAP kinase in arresting cell cycle at various stages in oocytes of different animal species is discussed.