Nitrogen (N) is a key nutrient that limits global primary productivity; hence, N-use efficiency is of compelling interest in agriculture and aquaculture. We used Chlamydomonas reinhardtii as a reference organism for a multicomponent analysis of the N starvation response. In the presence of acetate, respiratory metabolism is prioritized over photosynthesis; consequently, the N-sparing response targets proteins, pigments, and RNAs involved in photosynthesis and chloroplast function over those involved in respiration. Transcripts and proteins of the Calvin-Benson cycle are reduced in N-deficient cells, resulting in the accumulation of cycle metabolic intermediates. Both cytosolic and chloroplast ribosomes are reduced, but via different mechanisms, reflected by rapid changes in abundance of RNAs encoding chloroplast ribosomal proteins but not cytosolic ones. RNAs encoding transporters and enzymes for metabolizing alternative N sources increase in abundance, as is appropriate for the soil environmental niche of C. reinhardtii. Comparison of the N-replete versus N-deplete proteome indicated that abundant proteins with a high N content are reduced in N-starved cells, while the proteins that are increased have lower than average N contents. This sparing mechanism contributes to a lower cellular N/C ratio and suggests an approach for engineering increased N-use efficiency.

Many differentiated animal cells, and all higher plant cells, build interphase microtubule arrays of specific architectures without benefit of a central organizer, such as a centrosome, to control the location and geometry of microtubule nucleation. These acentrosomal arrays support essential cell functions such as morphogenesis [1, 2], but the mechanisms by which the new microtubules are positioned and oriented are poorly understood. In higher plants, nucleation of microtubules arises from distributed gamma-tubulin ring complexes (gamma-TuRCs) at the cell cortex that are associated primarily with existing microtubules [3-5] and from which new microtubules are nucleated in a geometrically bimodal fashion, either in parallel to the mother microtubule or as a branching event at a mean angle of approximately 40 degrees to the mother microtubule. By imaging the dynamics of individual nucleation events in Arabidopsis, we found that a conserved peripheral protein of the gamma-TuRC, GCP-WD/NEDD1 [6-8], associated with motile gamma-TuRCs and localized to nucleation events. Knockdown of this essential protein resulted in reduction of gamma-TuRC recruitment to cortical microtubules and total nucleation frequency, showing that GCP-WD controls gamma-TuRC positioning and function in these interphase arrays. Further, we discovered an unexpected role for GCP-WD in determining the geometry of microtubule-dependent microtubule nucleation, where it acts to increase the likelihood of branching over parallel nucleation. Cells with normally complex patterns of cortical array organization constructed simpler arrays with cell-wide ordering, suggesting that control of nucleation frequency, positioning, and geometry by GCP-WD allows plant cells to build alternative cortical array architectures.

Chlamydomonas reinhardtii (Chlamydomonas throughout) is a metabolically versatile, soil-dwelling photosynthetic alga, capable of modulating its metabolism as environmental conditions change; this flexibility enables sustained energy generation and maintenance of cellular redox balance. Because of its many unique features, Chlamydomonas has become a model organism for understanding a range of biological, cellular, molecular, and physiological processes, including fermentation metabolism, which occur in both photosynthetic and non-photosynthetic organisms. Genes encoding proteins associated with multiple fermentation pathways have been identified on the Chlamydomonas genome. Controlled regulation of these pathways has allowed this alga to satisfy its energy requirements when O-2 levels become low, and studies of Chlamydomonas mutant strains have demonstrated substantial metabolic flexibility in the use of the various available fermentation pathways. Furthermore, when Chlamydomonas ferments polysaccharides under anoxic conditions, it has the ability to eliminate reducing power (which sustains glycolysis) through the production of H-2, a molecule that can be developed as a source of sustainable energy. H-2 is produced by three different pathways; direct biophotolysis, indirect biophotolysis, and dark fermentative metabolism. To date, with respect to the algae there is little known about the specific role(s) of the different branches of fermentation metabolism, how they sense oxic conditions, and the mechanisms involved in controlling these responses, at both the transcriptional and posttranscriptional levels. More is known about acclimation to anoxic conditions in vascular plants, and recently it was demonstrated that the responses of plants to anoxia depend upon specific ethylene response factor transcriptional elements that are destabilized under oxygen-replete conditions (Trends Plant Sci 17: 129-138, 2012; Curr Opin Plant Biol 13: 489-494, 2010). In the present review, we focus on fermentation metabolism in Chlamydomonas and other green algae, with some discussion of plants when relevant. We also explore different methods that can be used to probe the physiology of cells exposed to hypoxic/anoxic conditions.

The microtubule plus-end tracking proteins (+TIPs) END BINDING1b (EB1b) and SPIRAL1 (SPR1) are required for normal cell expansion and organ growth. EB proteins are viewed as central regulators of +TIPs and cell polarity in animals; SPR1 homologs are specific to plants. To explore if EB1b and SPR1 fundamentally function together, we combined genetic, biochemical, and cell imaging approaches in Arabidopsis thaliana. We found that eb1b-2 spr1-6 double mutant roots exhibit substantially more severe polar expansion defects than either single mutant, undergoing right-looping growth and severe axial twisting instead of waving on tilted hard-agar surfaces. Protein interaction assays revealed that EB1b and SPR1 bind each other and tubulin heterodimers, which is suggestive of a microtubule loading mechanism. EB1b and SPR1 show antagonistic association with microtubules in vitro. Surprisingly, our combined analyses revealed that SPR1 can load onto microtubules and function independently of EB1 proteins, setting SPR1 apart from most studied +TIPs in animals and fungi. Moreover, we found that the severity of defects in microtubule dynamics in spr1 eb1b mutant hypocotyl cells correlated well with the severity of growth defects. These data indicate that SPR1 and EB1b have complex interactions as they load onto microtubule plus ends and direct polar cell expansion and organ growth in response to directional cues.

Because algae have become more accepted as sources of human nutrition, phylogenetic analysis can help resolve the taxonomy of taxa that have not been well studied. This can help establish algal evolutionary relationships. Here, we compare Auxenochlorella protothecoides and 23 strains of Prototheca based on their complete 16S and partial 23S plastid rDNA sequences along with nutrient utilization (auxanographic) profiles. These data demonstrate that some of the species groupings are not in agreement with the molecular phylogenetic analyses and that auxanographic profiles are poor predictors of phylogenetic relationships.

Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. We monitored the distribution and mobility of fluorescently labeled cellulose synthases (CESAs) in live Arabidopsis cells under chemical exposure to characterize their subcellular effects. CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. KOR1 accumulated in bodies similar to CESA; however, POM2/CSI1 dissociated into the cytoplasm. In addition, MT stability was altered without direct inhibition of MT polymerization, suggesting a feedback mechanism caused by cellulose interference. The selectivity of CESTRIN was assessed using a variety of subcellular markers for which no morphological effect was observed. The association of CESAs with vesicles decorated by the trans-Golgi network-localized protein SYNTAXIN OF PLANTS61 (SYP61) was increased under CESTRIN treatment, implicating SYP61 compartments in CESA trafficking. The properties of CESTRIN compared with known CESA inhibitors afford unique avenues to study and understand the mechanism under which PM-associated CSCs are maintained and interact with MTs and to dissect their trafficking routes in etiolated hypocotyls.

The green alga Chlamydomonas reinhardtii is a popular unicellular organism for studying photosynthesis, cilia biogenesis, and micronutrient homeostasis. Ten years since its genome project was initiated an iterative process of improvements to the genome and gene predictions has propelled this organism to the forefront of the omics era. Housed at Phytozome, the plant genomics portal of the Joint Genome Institute (JGI), the most up-to-date genomic data include a genome arranged on chromosomes and high-quality gene models with alternative splice forms supported by an abundance of whole transcriptome sequencing (RNA-Seq) data. We present here the past, present, and future of Chlamydomonas genomics. Specifically, we detail progress on genome assembly and gene model refinement, discuss resources for gene annotations, functional predictions, and locus ID mapping between versions and, importantly, outline a standardized framework for naming genes.

Reorganization of the cortical microtubule cytoskeleton is critical for guard cell function. Here, we investigate how environmental and hormonal signals cause these rearrangements and find that COP1, a RING-finger-type ubiquitin E3 ligase, is required for degradation of tubulin, likely by the 26S proteasome. This degradation is required for stomatal closing. In addition to regulating the cytoskeleton, we show that cop1 mutation impaired the activity of S-type anion channels, which are critical for stomatal closure. Thus, COP1 is revealed as a potential coordinator of cytoskeletal and electrophysiological activities required for guard cell function.

Chlamydomonas reinhardtii is well adapted to survive under different environmental conditions due to the unique flexibility of its metabolism. Here we report metabolic pathways that are active during acclimation to anoxia, but were previously not thoroughly studied under dark, anoxic H2-producing conditions in this model green alga. Proteomic analyses, using 2D-differential in-gel electrophoresis in combination with shotgun mass fingerprinting, revealed increased levels of proteins involved in the glycolytic pathway downstream of 3-phosphoglycerate, the glyoxylate pathway, and steps of the tricarboxylic acid (TCA) reactions. Upregulation of the enzyme, isocitrate lyase (ICL), was observed, which was accompanied by increased intracellular succinate levels, suggesting the functioning of glyoxylate pathway reactions. The ICL-inhibitor study revealed presence of reverse TCA reactions under these conditions. Contributions of the serine-isocitrate lyase pathway, glycine cleavage system, and c1-THF/serine hydroxymethyltransferase pathway in the acclimation to dark anoxia were found. We also observed increased levels of amino acids (AAs) suggesting nitrogen reorganization in the form of de novo AA biosynthesis during anoxia. Overall, novel routes for reductant utilization, in combination with redistribution of carbon and nitrogen, are used by this alga during acclimation to O-2 deprivation in the dark.

Microtubule nucleation in interphase plant cells primarily occurs through branching from pre-existing microtubules at dispersed sites in the cell cortex(1,2,3). The minus ends of new microtubules are often released from the sites of nucleation, and the free microtubules are then transported to new locations by polymer treadmilling(1). These nucleation-and-release events are characteristic features of plant arrays in interphase cells, but little is known about the spatiotemporal control of these events by nucleating protein complexes. We visualized the dynamics of two fluorescently-tagged gamma-tubulin complex proteins, GCP2 and GCP3, in Arabidopsis thaliana. These probes labelled motile complexes in the cytosol that transiently stabilized at fixed locations in the cell cortex. Recruitment of labelled complexes occurred preferentially along existing cortical microtubules, from which new microtubule was synthesized in a branching manner, or in parallel to the existing microtubule. Complexes localized to microtubules were approximately 10-fold more likely to display nucleation than were complexes recruited to other locations. Nucleating complexes remained stable until daughter microtubules were either completely depolymerized from their plus ends or released by katanin-dependent severing activity. These observations suggest that the nucleation complexes are primarily activated on association with microtubule lattices, and that nucleation complex stability depends on association with daughter microtubules and is regulated in part by katanin activity.