AGJM21PhMTS Illumina's proprietary software was used for basecalling.The reads were aligned using bowtie in single-end mode and with a maximum tolerance of 3 mismatches to the Au10.2 transcripts sequences (http://www.phytozome.net/chlamy), corresponding to the version 4.0 assembly of the Chlamydomonas genome.Expression estimates were obtained for each individual run in units of RPKMs (reads per kilobase of mappable transcript length per million mapped reads) after normalization by the number of aligned reads and transcript mappable length.Genome_build: version 4.0 assembly of the Chlamydomonas genome (http://genome.jgi-psf.org/Chlre4/Chlre4.home.html)Supplementary_files_format_and_content: Processed files correpond to average expression estimates per experiment in units of RPKMs.

Chlamydomonas reinhardtii insertion mutants disrupted for genes encoding acetate kinases (EC 2.7.2.1) (ACK1 and ACK2) and a phosphate acetyltransferase (EC 2.3.1.8) (PAT2, but not PAT1) were isolated to characterize fermentative acetate production. ACK1 and PAT2 were localized to chloroplasts, while ACK2 and PAT1 were shown to be in mitochondria. Characterization of the mutants showed that PAT2 and ACK1 activity in chloroplasts plays a dominant role (relative to ACK2 and PAT1 in mitochondria) in producing acetate under dark, anoxic conditions and, surprisingly, also suggested that Chlamydomonas has other pathways that generate acetate in the absence of ACK activity. We identified a number of proteins associated with alternative pathways for acetate production that are encoded on the Chlamydomonas genome. Furthermore, we observed that only modest alterations in the accumulation of fermentative products occurred in the ack1, ack2, and ack1 ack2 mutants, which contrasts with the substantial metabolite alterations described in strains devoid of other key fermentation enzymes.

Central to the building and reorganizing cytoskeletal arrays is creation of new polymers. Although nucleation has been the major focus of study for microtubule generation, severing has been proposed as an alternative mechanism to create new polymers, a mechanism recently shown to drive the reorientation of cortical arrays of higher plants in response to blue light perception. Severing produces new plus ends behind the stabilizing GTP-cap. An important and unanswered question is how these ends are stabilized in vivo to promote net microtubule generation. Here we identify the conserved protein CLASP as a potent stabilizer of new plus ends created by katanin severing in plant cells. Clasp mutants are defective in cortical array reorientation. In these mutants, both rescue of shrinking plus ends and the stabilization of plus ends immediately after severing are reduced. Computational modeling reveals that it is the specific stabilization of severed ends that best explains CLASP's function in promoting microtubule amplification by severing and array reorientation.

Symbiosis between unicellular dinoflagellates (genus Symbiodinium) and their cnidarian hosts (e.g. corals, sea anemones) is the foundation of coral reef ecosystems. Dysfunction of this symbiosis under changing environmental conditions has led to global reef decline. Little information is known about Symbiodinium gene expression and mechanisms by which light impacts host-symbiont associations. To address these issues, we generated a transcriptome from axenic Symbiodinium strain SSB01. Here we report features of the transcriptome, including occurrence and length distribution of spliced leader sequences, the functional landscape of encoded proteins and the impact of light on gene expression. Expression of many Symbiodinium genes appears to be significantly impacted by light. Transcript encoding cryptochrome 2 declined in high light while some transcripts for Regulators of Chromatin Condensation (RCC1) declined in the dark. We also identified a transcript encoding a light harvesting AcpPC protein with homology to Chlamydomonas LHCSR2. The level of this transcript increased in high light autotrophic conditions, suggesting that it is involved in photo-protection and the dissipation of excess absorbed light energy. The most extensive changes in transcript abundances occurred when the algae were transferred from low light to darkness. Interestingly, transcripts encoding several cell adhesion proteins rapidly declined following movement of cultures to the dark, which correlated with a dramatic change in cell surface morphology, likely reflecting the complexity of the extracellular matrix. Thus, light-sensitive cell adhesion proteins may play a role in establishing surface architecture, which may in turn alter interactions between the endosymbiont and its host.

Enormous societal challenges, such as feeding and providing energy for a growing population in a dramatically changing climate, necessitate technological advances in plant science. Plant cells are fundamental organizational units that mediate the production, transport, and storage of our primary food sources, and they sequester a significant proportion of the world's carbon. New technologies allow comprehensive descriptions of cells that could accelerate research across fields of plant science. Complementary to the efforts towards understanding the cellular diversity in humanbrain and immune systems, a Plant Cell Atlas (PCA) that maps molecular machineries to cellular and subcellular domains, follows their dynamic movements, and describes their interactions would accelerate discovery in plant science and help to solve imminent societal problems.

Chlamydomonas reinhardtii is a unicellular, soil-dwelling (and aquatic) green alga that has significant metabolic flexibility for balancing redox equivalents and generating ATP when it experiences hypoxic/anoxic conditions. The diversity of pathways available to ferment sugars is often revealed in mutants in which the activities of specific branches of fermentative metabolism have been eliminated; compensatory pathways that have little activity in parental strains under standard laboratory fermentative conditions are often activated. The ways in which these pathways are regulated and integrated have not been extensively explored. In this review, we primarily discuss the intricacies of dark anoxic metabolism in Chlamydomonas, but also discuss aspects of dark oxic metabolism, the utilization of acetate, and the relatively uncharacterized but critical interactions that link chloroplastic and mitochondrial metabolic networks.

The cortical microtubule arrays of higher plants are organized without centrosomes and feature treadmilling polymers that are dynamic at both ends. The control of polymer end stability is fundamental for the assembly and organization of cytoskeletal arrays, yet relatively little is understood about how microtubule minus ends are controlled in acentrosomal microtubule arrays, and no factors have been identified that act at the treadmilling minus ends in higher plants. Here, we identify Arabidopsis thaliana SPI RAL2 (SPR2) as a protein that tracks minus ends and protects them against subunit loss. SPR2 function is required to facilitate the rapid reorientation of plant cortical arrays as stimulated by light perception, a process that is driven by microtubule severing to create a new population of microtubules. Quantitative live-cell imaging and computer simulations reveal that minus protection by SPR2 acts by an unexpected mechanism to promote the lifetime of potential SPR2 severing sites, increasing the likelihood of severing and thus the rapid amplification of the new microtubule array.

Phosphorus (P) is an essential nutrient that is integral to lipids, nucleic acids and various metabolites, and also binds proteins covalently in ways that may alter their catalytic activities and interactions with other proteins. Phosphate (PO43-), both inorganic and organic, is the major source of P for nearly all microbes, algae and plants, although in some environments organic PO43- molecules (including nucleic acids, phospholipids and phosphonates) comprise a significant proportion of the available P. Many natural environments have low levels of available P, which limits the growth of plants, algae and microbes. These organisms have developed a diversity of strategies to scavenge PO43- from external sources, to recycle and balance P utilization within the cell in response to environmental conditions, and to coordinate cell growth and division with P availability. Furthermore, agricultural lands may be depleted of many nutrients including nitrogen (N) and P. Fertilizers with high N and P contents are liberally applied to many millions of acres of farmland globally, and these could be leached from the soil to contaminate lakes, rivers, ponds and coastal waters. Research groups are now starting to understand the complexity of P cycling in the environment and the molecular mechanisms associated with the acclimatisation of photoautotrophic and heterotrophic organisms to P limitation. In this chapter, P availability in the natural environment is discussed, and the physiological and molecular strategies used by algae (in the context of other organisms) for the efficient capture of external P, to recycle P-containing molecules in the cell, and to reconfigure cellular metabolism to sustain viability in a P-limited landscape, are emphasised.