3-D Ultrastructure of O. tauri: Electron Cryotomography of an Entire Eukaryotic Cell

The hallmark of eukaryotic cells is their segregation of key biological functions into discrete, membrane-bound organelles. Creating accurate models of their ultrastructural complexity has been difficult in part because of the limited resolution of light microscopy and the artifact-prone nature of conventional electron microscopy. Here we explored the potential of the emerging technology electron cryotomography to produce three-dimensional images of an entire eukaryotic cell in a near-native state. Ostreococcus tauri was chosen as the specimen because as a unicellular picoplankton with just one copy of each organelle, it is the smallest known eukaryote and was therefore likely to yield the highest resolution images. Whole cells were imaged at various stages of the cell cycle, yielding 3-D reconstructions of complete chloroplasts, mitochondria, endoplasmic reticula, Golgi bodies, peroxisomes, microtubules, and putative ribosome distributions in-situ. Surprisingly, the nucleus was seen to open long before mitosis, and while one microtubule (or two in some predivisional cells) was consistently present, no mitotic spindle was ever observed, prompting speculation that a single microtubule might be sufficient to segregate multiple chromosomes

Chlorophyll-deficient mutants of Chlamydomonas reinhardtii that accumulate magnesium protoporphyrin IX

Two Chlamydomonas reinhardtii mutants defective in CHLM encoding Mg-protoporphyrin IX methyltransferase (MgPMT) were identified. The mutants, one with a missense mutation (chlM-1) and a second mutant with a splicing defect (chlM-2), do not accumulate chlorophyll, are yellow in the dark and dim light, and their growth is inhibited at higher light intensities. They accumulate Mg-protoporphyrin IX (MgProto), the substrate of MgPMT and this may be the cause for their light sensitivity. In the dark, both mutants showed a drastic reduction in the amounts of core proteins of photosystems I and II and light-harvesting chlorophyll a/b-binding proteins. However, LHC mRNAs accumulated above wild-type levels. The accumulation of the transcripts of the LHC and other genes that were expressed at higher levels in the mutants during dark incubation was attenuated in the initial phase of light exposure. No regulatory effects of the constitutively 7- to 18-fold increased MgProto levels on gene expression were detected, supporting previous results in which MgProto and heme in Chlamydomonas were assigned roles as second messengers only in the transient activation of genes by light

Chlorophylls, ligands and assembly of light-harvesting complexes in chloroplasts

Chlorophyll (Chl) b serves an essential function in accumulation of light-harvesting complexes (LHCs) in plants. In this article, this role of Chl b is explored by considering the properties of Chls and the ligands with which they interact in the complexes. The overall properties of the Chls, not only their spectral features, are altered as consequences of chemical modifications on the periphery of the molecules. Important modifications are introduction of oxygen atoms at specific locations and reduction or desaturation of sidechains. These modifications influence formation of coordination bonds by which the central Mg atom, the Lewis acid, of Chl molecules interacts with amino acid sidechains, as the Lewis base, in proteins. Chl a is a versatile Lewis acid and interacts principally with imidazole groups but also with sidechain amides and water. The 7-formyl group on Chl b withdraws electron density toward the periphery of the molecule and consequently the positive Mg is less shielded by the molecular electron cloud than in Chl a. Chl b thus tends to form electrostatic bonds with Lewis bases with a fixed dipole, such as water and, in particular, peptide backbone carbonyl groups. The coordination bonds are enhanced by H-bonds between the protein and the 7-formyl group. These additional strong interactions with Chl b are necessary to achieve assembly of stable LHCs

Ion homeostasis in the Chloroplast

peer reviewedThe chloroplast is an organelle of high demand for macro- and micro-nutrient ions, which are required for the maintenance of the photosynthetic process. To avoid deficiency while preventing excess, homeostasis mechanisms must be tightly regulated. Here, we describe the needs for nutrient ions in the chloroplast and briefly highlight their functions in the chloroplastidial metabolism. We further discuss the impact of nutrient deficiency on chloroplasts and the acclimation mechanisms that evolved to preserve the photosynthetic apparatus. We finally present what is known about import and export mechanisms for these ions. Whenever possible, a comparison between cyanobacteria, algae and plants is provided to add an evolutionary perspective to the description of ion homeostasis mechanisms in photosynthesis

Loss of the SPHF homologue Slr1768 leads to a catastrophic failure in the maintenance of thylakoid membranes in synechocystis sp. PCC 6803

Background: In cyanobacteria the photosystems are localised to, and maintained in, specialist membranes called the thylakoids. The mechanism driving the biogenesis of the thylakoid membranes is still an open question, with only two potential biogenesis factors, Vipp1 and Alb3 currently identified. Methodology/Principal Findings: We generated a slr1768 knockout using the pGEM T-easy vector and REDIRECT. By comparing growth and pigment content (chlorophyll a fluoresence) of the Delta slr1768 mutant with the wild-type, we found that Dslr1768 has a conditional phenotype; specifically under high light conditions (130 mu mol m(-2) s(-1)) thylakoid biogenesis is disrupted leading to cell death on a scale of days. The thylakoids show considerable disruption, with loss of both structure and density, while chlorophyll a density decreases with the loss of thylakoids, although photosynthetic efficiency is unaffected. Under low light (30 mu mol m(-2) s(-1)) the phenotype is significantly reduced, with a growth rate similar to the wildtype and only a low frequency of cells with evident thylakoid disruption. Conclusions/Significance: This is the first example of a gene that affects the maintenance of the thylakoid membranes specifically under high light, and which displays a phenotype dependent on light intensity. Our results demonstrate that Slr1768 has a leading role in acclimatisation, linking light damage with maintenance of the thylakoids

Thylakoid membrane perforations and connectivity enable intracellular traffic in cyanobacteria

Cyanobacteria, the progenitors of plant and algal chloroplasts, enabled aerobic life on earth by introducing oxygenic photosynthesis. In most cyanobacteria, the photosynthetic membranes are arranged in multiple, seemingly disconnected, concentric shells. In such an arrangement, it is unclear how intracellular trafficking proceeds and how different layers of the photosynthetic membranes communicate with each other to maintain photosynthetic homeostasis. Using electron microscope tomography, we show that the photosynthetic membranes of two distantly related cyanobacterial species contain multiple perforations. These perforations, which are filled with particles of different sizes including ribosomes, glycogen granules and lipid bodies, allow for traffic throughout the cell. In addition, different layers of the photosynthetic membranes are joined together by internal bridges formed by branching and fusion of the membranes. The result is a highly connected network, similar to that of higher-plant chloroplasts, allowing water-soluble and lipid-soluble molecules to diffuse through the entire membrane network. Notably, we observed intracellular membrane-bounded vesicles, which were frequently fused to the photosynthetic membranes and may play a role in transport to these membranes