Insights into the Evolution of Multicellularity from the Sea Lettuce Genome.

We report here the 98.5 Mbp haploid genome (12,924 protein coding genes) of Ulva mutabilis, a ubiquitous and iconic representative of the Ulvophyceae or green seaweeds. Ulva's rapid and abundant growth makes it a key contributor to coastal biogeochemical cycles; its role in marine sulfur cycles is particularly important because it produces high levels of dimethylsulfoniopropionate (DMSP), the main precursor of volatile dimethyl sulfide (DMS). Rapid growth makes Ulva attractive biomass feedstock but also increasingly a driver of nuisance "green tides." Ulvophytes are key to understanding the evolution of multicellularity in the green lineage, and Ulva morphogenesis is dependent on bacterial signals, making it an important species with which to study cross-kingdom communication. Our sequenced genome informs these aspects of ulvophyte cell biology, physiology, and ecology. Gene family expansions associated with multicellularity are distinct from those of freshwater algae. Candidate genes, including some that arose following horizontal gene transfer from chromalveolates, are present for the transport and metabolism of DMSP. The Ulva genome offers, therefore, new opportunities to understand coastal and marine ecosystems and the fundamental evolution of the green lineage.

Comparative phosphoproteomics to identify targets of the clock-relevant casein kinase 1 in C. reinhardtii Flagella.

In the green biflagellate alga Chlamydomonas reinhardtii different clock-relevant components have been identified that are involved in maintaining phase, period, and amplitude of circadian rhythms. It became evident that several of them are interconnected to flagellar function such as CASEIN KINASE1 (CK1). CK1 is involved in keeping the period. But it is also relevant for the formation of flagella, where it is physically located, and it controls the swimming velocity. In this chapter, we describe (1) how the flagellar sub-proteome is purified, (2) how phosphopeptides from this organelle are enriched, (3) how in vivo phosphorylation sites are determined, and (4) how direct and indirect flagellar targets of CK1 can be found using a specific inhibitor. Such a procedure can also be employed with other clock-relevant kinases if specific inhibitors or mutants are available.

The heme-binding protein SOUL3 of Chlamydomonas reinhardtii influences size and position of the eyespot.

The flagellated green alga Chlamydomonas reinhardtii has a primitive visual system, the eyespot. It is situated at the cells equator and allows the cell to phototax. In a previous proteomic analysis of the eyespot, the SOUL3 protein was identified among 202 proteins. Here, we investigate the properties and functions of SOUL3. Heterologously expressed SOUL3 is able to bind specifically to hemin. In C. reinhardtii, SOUL3 is expressed at a constant level over the diurnal cycle, but forms protein complexes that differ in size during day and night phases. SOUL3 is primarily localized in the eyespot and it is situated in the pigment globule layer thereof. This is in contrast to the channelrhodopsin photoreceptors, which are localized in the plasma membrane region of the eyespot. Knockdown lines with a significantly reduced SOUL3 level are characterized by mislocalized eyespots, a decreased eyespot size, and alterations in phototactic behavior. Mislocalizations were either anterior or posterior and did not affect association with acetylated microtubules of the daughter four-membered rootlet. Our data suggest that SOUL3 is involved in the organization and placement of the eyespot within the cell.

Application of phosphoproteomics to find targets of casein kinase 1 in the flagellum of chlamydomonas.

The green biflagellate alga Chlamydomonas reinhardtii serves as model for studying structural and functional features of flagella. The axoneme of C. reinhardtii anchors a network of kinases and phosphatases that control motility. One of them, Casein Kinase 1 (CK1), is known to phosphorylate the Inner Dynein Arm I1 Intermediate Chain 138 (IC138), thereby regulating motility. CK1 is also involved in regulating the circadian rhythm of phototaxis and is relevant for the formation of flagella. By a comparative phosphoproteome approach, we determined phosphoproteins in the flagellum that are targets of CK1. Thereby, we applied the specific CK1 inhibitor CKI-7 that causes significant changes in the flagellum phosphoproteome and reduces the swimming velocity of the cells. In the CKI-7-treated cells, 14 phosphoproteins were missing compared to the phosphoproteome of untreated cells, including IC138, and four additional phosphoproteins had a reduced number of phosphorylation sites. Notably, inhibition of CK1 causes also novel phosphorylation events, indicating that it is part of a kinase network. Among them, Glycogen Synthase Kinase 3 is of special interest, because it is involved in the phosphorylation of key clock components in flies and mammals and in parallel plays an important role in the regulation of assembly in the flagellum.

Analysis of flagellar phosphoproteins from Chlamydomonas reinhardtii.

Cilia and flagella are cell organelles that are highly conserved throughout evolution. For many years, the green biflagellate alga Chlamydomonas reinhardtii has served as a model for examination of the structure and function of its flagella, which are similar to certain mammalian cilia. Proteome analysis revealed the presence of several kinases and protein phosphatases in these organelles. Reversible protein phosphorylation can control ciliary beating, motility, signaling, length, and assembly. Despite the importance of this posttranslational modification, the identities of many ciliary phosphoproteins and knowledge about their in vivo phosphorylation sites are still missing. Here we used immobilized metal affinity chromatography to enrich phosphopeptides from purified flagella and analyzed them by mass spectrometry. One hundred forty-one phosphorylated peptides were identified, belonging to 32 flagellar proteins. Thereby, 126 in vivo phosphorylation sites were determined. The flagellar phosphoproteome includes different structural and motor proteins, kinases, proteins with protein interaction domains, and many proteins whose functions are still unknown. In several cases, a dynamic phosphorylation pattern and clustering of phosphorylation sites were found, indicating a complex physiological status and specific control by reversible protein phosphorylation in the flagellum.

Sub-proteome analysis in the green flagellate alga Chlamydomonas reinhardtii.

In the past years, research on the flagellate unicellular alga Chlamydomonas reinhardtii has entered a new era based on the availability of its complete genome. Since this green alga can be grown relatively easy in a short time-range, sufficient biological material is available to efficiently establish biochemical purification procedures of sub-cellular fractions. Combined with the available genome sequences, this paved the way to perform analysis of specific sub-proteomes by mass spectrometry. In this review, several approaches that provided comprehensive lists of components of certain sub-cellular compartments and their biological relevance will be described. These include proteins of chloroplast ribosomes, of flagella, of the eyespot as well as posttranslational and environmentally modified sub-proteomes. The power of such proteome approaches lies in the identification of novel components and modifications of a given sub-proteome that have not been discovered before. Information is usually gained at a large scale and is very valuable to further understand biological processes of a given cellular sub-compartment. But clearly the arduous task has then to be performed to further analyze the function of specific proteins/genes by RNA interference technology, mutant analyses or methods for identifying the protein interaction network within a sub-proteome.