Eukaryotic microalgae hold great promise for the bioproduction of fuels and higher value chemicals. However, compared with model genetic organisms such asEscherichia coliandSaccharomyces cerevisiae, characterization of the complex biology and biochemistry of algae and strain improvement has been hampered by the inefficient genetic tools. To date, many algal species are transformable only via particle bombardment, and the introduced DNA is integrated randomly into the nuclear genome. Here we describe the first nuclear episomal vector for diatoms and a plasmid delivery method via conjugation fromEscherichia colito the diatomsPhaeodactylum tricornutumandThalassiosira pseudonana. We identify a yeast-derived sequence that enables stable episome replication in these diatoms even in the absence of antibiotic selection and show that episomes are maintained as closed circles at copy number equivalent to native chromosomes. This highly efficient genetic system facilitates high-throughput functional characterization of algal genes and accelerates molecular phytoplankton research.

Environmental fluctuations affect distribution, growth and abundance of diatoms in nature, with iron (Fe) availability playing a central role. Studies on the response of diatoms to low Fe have either utilized continuous (24 hr) illumination or sampled a single time of day, missing any temporal dynamics. We profiled the physiology, metabolite composition, and global transcripts of the pennate diatom Phaeodactylum tricornutum during steady-state growth at low, intermediate, and high levels of dissolved Fe over light:dark cycles, to better understand fundamental aspects of genetic control of physiological acclimation to growth under Fe-limitation. We greatly expand the catalog of genes involved in the low Fe response, highlighting the importance of intracellular trafficking in Fe-limited diatoms. P. tricornutum exhibited transcriptomic hallmarks of slowed growth leading to prolonged periods of cell division/silica deposition, which could impact biogeochemical carbon sequestration in Fe-limited regions. Light harvesting and ribosome biogenesis transcripts were generally reduced under low Fe while transcript levels for genes putatively involved in the acquisition and recycling of Fe were increased. We also noted shifts in expression towards increased synthesis and catabolism of branched chain amino acids in P. tricornutum grown at low Fe whereas expression of genes involved in central core metabolism were relatively unaffected, indicating that essential cellular function is protected. Beyond the response of P. tricornutum to low Fe, we observed major coordinated shifts in transcript control of primary and intermediate metabolism over light:dark cycles which contribute to a new view of the significance of distinctive diatom pathways, such as mitochondrial glycolysis and the ornithine-urea cycle. This study provides new insight into transcriptional modulation of diatom physiology and metabolism across light:dark cycles in response to Fe availability, providing mechanistic understanding for the ability of diatoms to remain metabolically poised to respond quickly to Fe input and revealing strategies underlying their ecological success. Oceanic diatoms live in constantly fluctuating environments to which they must adapt in order to survive. During sunlit hours, photosynthesis occurs allowing diatoms to store energy used at night to sustain energy demands. Cellular and molecular mechanisms for regulation of phytoplankton growth are important to understand because of their environmental roles at the base of food webs and in regulating carbon flux out of the atmosphere. In ocean ecosystems, the availability of iron (Fe) commonly limits phytoplankton growth and diatoms typically outcompete other phytoplankton when Fe is added, indicating they have adaptations allowing them to both survive at low Fe and rapidly respond to Fe additions. These adaptations may be unique depending on isolation from coastal or oceanic locations. To identify adaptive strategies, we characterized the response of a model diatom, Phaeodactylum tricornutum, to limiting Fe conditions over day:night cycles using a combination of gene expression analyses, metabolite, and physiology measurements. Major coordinated shifts in metabolism and growth were documented over diel cycles, with peak expression of low Fe expressed genes in the dark phase. Diatoms respond to limiting Fe by increasing Fe acquisition, while decreasing growth rate through slowed cell cycle progression, reduced energy acquisition, and subtle metabolic remodeling.

Despite the growing interest in diatom genomics, detailed time series of gene expression in relation to key cellular processes are still lacking. Here, we investigated the relationships between the cell cycle and chloroplast development in the pennate diatom Seminavis robusta. This diatom possesses two chloroplasts with a well-orchestrated developmental cycle, common to many pennate diatoms. By assessing the effects of induced cell cycle arrest with microscopy and flow cytometry, we found that division and reorganization of the chloroplasts are initiated only after S-phase progression. Next, we quantified the expression of the S. robusta FtsZ homolog to address the division status of chloroplasts during synchronized growth and monitored microscopically their dynamics in relation to nuclear division and silicon deposition. We show that chloroplasts divide and relocate during the S/G2 phase, after which a girdle band is deposited to accommodate cell growth. Synchronized cultures of two genotypes were subsequently used for a cDNA-amplified fragment length polymorphism-based genome-wide transcript profiling, in which 917 reproducibly modulated transcripts were identified. We observed that genes involved in pigment biosynthesis and coding for light-harvesting proteins were up-regulated during G2/M phase and cell separation. Light and cell cycle progression were both found to affect fucoxanthin-chlorophyll a/c-binding protein expression and accumulation of fucoxanthin cell content. Because chloroplasts elongate at the stage of cytokinesis, cell cycle-modulated photosynthetic gene expression and synthesis of pigments in concert with cell division might balance chloroplast growth, which confirms that chloroplast biogenesis in S. robusta is tightly regulated.

Background: Despite the enormous importance of diatoms in aquatic ecosystems and their broad industrial potential, little is known about their life cycle control. Diatoms typically inhabit rapidly changing and unstable environments, suggesting that cell cycle regulation in diatoms must have evolved to adequately integrate various environmental signals. The recent genome sequencing of Thalassiosira pseudonana and Phaeodactylum tricornutum allows us to explore the molecular conservation of cell cycle regulation in diatoms. Results: By profile-based annotation of cell cycle genes, counterparts of conserved as well as new regulators were identified in T. pseudonana and P. tricornutum. In particular, the cyclin gene family was found to be expanded extensively compared to that of other eukaryotes and a novel type of cyclins was discovered, the diatom-specific cyclins. We established a synchronization method for P. tricornutum that enabled assignment of the different annotated genes to specific cell cycle phase transitions. The diatom-specific cyclins are predominantly expressed at the G1-to-S transition and some respond to phosphate availability, hinting at a role in connecting cell division to environmental stimuli. Conclusion: The discovery of highly conserved and new cell cycle regulators suggests the evolution of unique control mechanisms for diatom cell division, probably contributing to their ability to adapt and survive under highly fluctuating environmental conditions.

Diatoms are highly abundant unicellular algae that often dominate pelagic as well as benthic primary production in the oceans and inland waters. Being strictly dependent on silica to build their biomineralized cell walls, marine diatoms precipitate 240 × 10¹² mol Si per year, which makes them the major sink in the global Si cycle. Dissolved silicic acid (dSi) availability frequently limits diatom productivity and influences species composition of communities. We show that benthic diatoms selectively perceive and behaviourally react to gradients of dSi. Cell speed increases under dSi-limited conditions in a chemokinetic response and, if gradients of this resource are present, increased directionality of cell movement promotes chemotaxis. The ability to exploit local and short-lived dSi hotspots using a specific search behaviour likely contributes to micro-scale patch dynamics in biofilm communities. On a global scale this behaviour might affect sediment–water dSi fluxes and biogeochemical cycling.