Nicoletta La Rocca

The Response of Nannochloropsis gaditana to Nitrogen Starvation Includes De Novo Biosynthesis of Triacylglycerols, a Decrease of Chloroplast Galactolipids, and Reorganization of the Photosynthetic Apparatus

ABSTRACT Microalgae of the genus Nannochloropsis are capable of accumulating triacylglycerols (TAGs) when exposed to nutrient limitation (in particular, nitrogen [N]) and are therefore considered promising organisms for biodiesel production. Here, after nitrogen removal from the medium, Nannochloropsis gaditana cells showed extensive triacylglycerol accumulation (38% TAG on a dry weight basis). Triacylglycerols accumulated during N deprivation harbored signatures, indicating that they mainly stemmed from freshly synthesized fatty acids, with a small proportion originating from a recycling of membrane glycerolipids. The amount of chloroplast galactoglycerolipids, which are essential for the integrity of thylakoids, decreased, while their fatty acid composition appeared to be unaltered. In starved cells, galactolipids were kept at a level sufficient to maintain chloroplast integrity, as confirmed by electron microscopy. Consistently, N-starved Nannochloropsis cells contained less photosynthetic membranes but were still efficiently performing photosynthesis. N starvation led to a modification of the photosynthetic apparatus with a change in pigment composition and a decrease in the content of all the major electron flow complexes, including photosystem II, photosystem I, and the cytochrome b6f complex. The photosystem II content was particularly affected, leading to the inhibition of linear electron flow from water to CO2. Such a reduction, however, was partially compensated for by activation of alternative electron pathways, such as cyclic electron transport. Overall, these changes allowed cells to modify their energetic metabolism in order to maintain photosynthetic growth.

Catalase Takes Part in Rat Liver Mitochondria Oxidative Stress Defense

Highly purified rat liver mitochondria (RLM) when exposed to tert-butylhydroperoxide undergo matrix swelling, membrane potential collapse, and oxidation of glutathione and pyridine nucleotides, all events attributable to the induction of mitochondrial permeability transition. Instead, RLM, if treated with the same or higher amounts of H2O2 or tyramine, are insensitive or only partially sensitive, respectively, to mitochondrial permeability transition. In addition, the block of respiration by antimycin A added to RLM respiring in state 4 conditions, or the addition of H2O2, results in O2 generation, which is blocked by the catalase inhibitors aminotriazole or KCN. In this regard, H2O2 decomposition yields molecular oxygen in a 2:1 stoichiometry, consistent with a catalatic mechanism with a rate constant of 0.0346 s-1. The rate of H2O2 consumption is not influenced by respiratory substrates, succinate or glutamate-malate, nor by N-ethylmaleimide, suggesting that cytochrome c oxidase and the glutathione-glutathione peroxidase system are not significantly involved in this process. Instead, H2O2 consumption is considerably inhibited by KCN or aminotriazole, indicating activity by a hemoprotein. All these observations are compatible with the presence of endogenous heme-containing catalase with an activity of 825 ± 15 units, which contributes to mitochondrial protection against endogenous or exogenous H2O2. Mitochondrial catalase in liver most probably represents regulatory control of bioenergetic metabolism, but it may also be proposed for new therapeutic strategies against liver diseases. The constitutive presence of catalase inside mitochondria is demonstrated by several methodological approaches as follows: biochemical fractionating, proteinase K sensitivity, and immunogold electron microscopy on isolated RLM and whole rat liver tissue.

Characterization and location of Src-dependent tyrosine phosphorylation in rat brain mitochondria

Analysis of protein phosphorylation in highly purified rat brain mitochondria revealed the presence of several alkali-stable phosphoproteins whose phosphorylation markedly increases upon treatment with peroxovanadate and Mn(2+), a property indicating tyrosine phosphorylation. These include three prominent bands, with apparent sizes of 50, 60, and 75 kDa, which are detectable by anti-phosphotyrosine. Tyrosine phosphorylation disappears when mitochondria are treated with PP2, an inhibitor of the Src kinase family, suggesting the presence of members of this family in rat brain mitochondria. Immunoblotting and immunoprecipitation assays of mitochondrial lysates confirmed the presence of Fyn, Src and Lyn kinases, as well as Csk, a protein kinase which negatively controls the activity of the Src kinase family. Results show that tyrosine-phosphorylated proteins are membrane-bound and that they are located on the inner surface of the outer membrane and/or the external surface of the inner membrane. Instead, Src tyrosine kinases are mainly located in the intermembrane space - in particular, as revealed by immunogold experiments for Lyn kinase, in the cristal lumen. Rat brain mitochondria were also found to possess a marked level of tyrosine phosphatase activity, strongly inhibited by peroxovanadate.

Thylakoid potassium channel is required for efficient photosynthesis in cyanobacteria

A potassium channel (SynK) of the cyanobacterium Synechocystis sp. PCC 6803, a photoheterotrophic model organism for the study of photosynthesis, has been recently identified and demonstrated to function as a potassium selective channel when expressed in a heterologous system and to be located predominantly to the thylakoid membrane in cyanobacteria. To study its physiological role, a SynK-less knockout mutant was generated and characterized. Fluorimetric experiments indicated that SynK-less cyanobacteria cannot build up a proton gradient as efficiently as WT organisms, suggesting that SynK might be involved in the regulation of the electric component of the proton motive force. Accordingly, measurements of flash-induced cytochrome b6f turnover and respiration pointed to a reduced generation of ΔpH and to an altered linear electron transport in mutant cells. The lack of the channel did not cause an altered membrane organization, but decreased growth and modified the photosystem II/photosystem I ratio at high light intensities because of enhanced photosensitivity. These data shed light on the function of a prokaryotic potassium channel and reports evidence, by means of a genetic approach, on the requirement of a thylakoid ion channel for optimal photosynthesis.

Modulation of mitochondrial K+ permeability and reactive oxygen species production by the p13 protein of human T-cell leukemia virus type 1

Human T-cell leukemia virus type-1 (HTLV-1) expresses an 87-amino acid protein named p13 that is targeted to the inner mitochondrial membrane. Previous studies showed that a synthetic peptide spanning an alpha helical domain of p13 alters mitochondrial membrane permeability to cations, resulting in swelling. The present study examined the effects of full-length p13 on isolated, energized mitochondria. Results demonstrated that p13 triggers an inward K(+) current that leads to mitochondrial swelling and confers a crescent-like morphology distinct from that caused by opening of the permeability transition pore. p13 also induces depolarization, with a matching increase in respiratory chain activity, and augments production of reactive oxygen species (ROS). These effects require an intact alpha helical domain and strictly depend on the presence of K(+) in the assay medium. The effects of p13 on ROS are mimicked by the K(+) ionophore valinomycin, while the protonophore FCCP decreases ROS, indicating that depolarization induced by K(+) vs. H(+) currents has different effects on mitochondrial ROS production, possibly because of their opposite effects on matrix pH (alkalinization and acidification, respectively). The downstream consequences of p13-induced mitochondrial K(+) permeability are likely to have an important influence on the redox state and turnover of HTLV-1-infected cells.

A Novel Potassium Channel in Photosynthetic Cyanobacteria

Elucidation of the structure-function relationship of a small number of prokaryotic ion channels characterized so far greatly contributed to our knowledge on basic mechanisms of ion conduction. We identified a new potassium channel (SynK) in the genome of the cyanobacterium Synechocystis sp. PCC6803, a photosynthetic model organism. SynK, when expressed in a K+-uptake-system deficient E.coli strain, was able to recover growth of these organisms. The protein functions as a potassium selective ion channel when expressed in Chinese Hamster Ovary cells. The location of SynK in cyanobacteria in both thylakoid and plasmamembranes was revealed by immunogold electron microscopy and Western blotting of isolated membrane fractions. SynK seems to be conserved during evolution, giving rise to a TPK (two-pore K+ channel) family member which is shown here to be located in the thylakoid membrane of Arabidopsis. Our work characterizes a novel cyanobacterial potassium channel and indicates the molecular nature of the first higher plant thylakoid cation channel, opening the way to functional studies.

Polyphasic approach and typification of selected Phormidium strains (Cyanobacteria)

Cyanobacteria (phylum Cyanophyta/Cyanobacteria, class Cyanophyceae) are among the most widespread organisms and are able to adapt themselves to different extreme environments. These micro‐organisms have an important ecological role, given their ability to perform oxygenic photosynthesis, and are employed in different fields based on their ability to produce several bioactive compounds. Their prokaryotic nature, the presence of many cryptic species, and the coexistence of different nomenclature systems make the taxonomic identification of cyanobacteria particularly difficult. Moreover, for several species, the original reference strains (holotypes) are lacking. Increasingly, authors are using a polyphasic approach to characterize cyanobacteria, while typification is a recent trend that is being used to solve the problem of missing holotypes in other micro‐organisms. Here we focus on a filamentous cyanobacterium, isolated from the Euganean Thermal District (Padova, Italy) and temporarily named strain ETS‐02, using a polyphasic approach that includes morphological, ultrastructural, biochemical (pigment and fatty acid content), physiological (nitrogen fixation), and genetic (16S rRNA, 16S–23S ITS, cpcB‐IGS‐cpcA, rpoC1, gyrB, rbcL, nifD loci) analyses. The description of Phormidium cf. irriguum CCALA 759 as the epitype of Phormidium irriguum was also used to complete the characterization of strain ETS‐02.

Grp94 is Tyr-phosphorylated by Fyn in the lumen of the endoplasmic reticulum and translocates to Golgi in differentiating myoblasts.

The endoplasmic-reticulum chaperone Grp94 is required for the cell surface export of molecules involved in the native immune response, in mesoderm induction and muscle development, but the signals responsible for Grp94 recruitment are still obscure. Here we show for the first time that Grp94 undergoes Tyr-phosphorylation in differentiating myogenic C2C12 cells. By means of phospho-proteomic and immunoprecipitation analyses, and the use of Src-specific inhibitors we demonstrate that the Src-tyrosine-kinase Fyn becomes active early after induction of C2C12 cell differentiation, in parallel with the recruitment and the Tyr-phosphorylation of Grp94, which peaks at 6-hour differentiation. Grp94 is Tyr-phosphorylated inside the endoplasmic reticulum by a lumenal Fyn, as indicated by fluorescence and electronmicroscopy immunolocalization, co-immunoprecipitation after chemical cross-linking and by treatment of intact endoplasmic-reticulum vesicles with proteinase K. Furthermore, fractionation of cellular membrane compartments and double-immunofluorescence studies showed that Tyr-phosphorylation of Grp94 is necessary for the protein translocation from the endoplasmic reticulum to the Golgi apparatus. These results indicate that Fyn-catalyzed Tyr-phosphorylation of Grp94 is an event required to promote the chaperone export from the endoplasmic reticulum occurring in the early phase of myoblast differentiation.

Pyramimonas australis sp. nov. (Prasinophyceae, Chlorophyta) from Antarctica : fine structure and molecular phylogeny

An undescribed marine Pyramimonas, P. australis Andreoli et Moro, sp. nov., forming a bloom in a hole of Terra Nova Bay (Ross Sea, Antarctica) sea ice, was collected, but could not be cultured. Consequently, the description of this new species is based on light and electron microscope observations on samples that were fixed or stored at −80  °C, and its phylogenetic position inferred from nuclear-encoded small-subunit ribosomal DNA (SSU rDNA) and chloroplast-encoded rbcL gene sequences. This is the third Antarctic species described for this genus. The ultrastructure of the cell is consistent with species of the subgenus Trichocystis McFadden, but differs in that it has unique body and cyst scales, and a different encystment procedure. The outermost layer of body scales is formed by flat box scales with peripheral perforations oriented parallel to the four edges and with a further eight central perforations oriented perpendicular to the peripheral ones. Crown scales, which in many other species of the genus form the outermost layer over the entire cell body, were observed in this species in the flagellar pit over the box scales. The flagella are covered by a pentagonal underlayer of scales and by limuloid scales with two subsidiary spines, in addition to the central one. Encystment begins in the flagellate form resulting in a cyst with an irregular wall bearing spine scales. Ultrastructural and molecular data confirm that P. australis belongs to the subgenus Trichocystis.

Polyphasic characterization of a thermo-tolerant filamentous cyanobacterium isolated from the Euganean thermal muds (Padua, Italy)

In this paper we report a morphological, ultrastructural, biochemical and molecular (16S rRNA, 16S–23S ITS, rbcL and rpoC1 gene sequencing) survey on a very thin, non-heterocystous, filamentous cyanobacterium, isolated from mats covering several mud maturation tanks of the Euganean Thermal District, at temperatures ranging from 26 to 59°C. Denaturing gradient gel electrophoresis results, obtained using cyanobacterial primers targeting the 16S rRNA gene, confirmed that this cyanobacterium is one of the commonest taxa growing in the mud tanks. Comparison with Geitlerinema sp. PCC 8501 (=Phormidium laminosum Gomont ex Gomont strain OH-1-p Cl 1), a thin thermobiotic species isolated from hot springs of Oregon and morphologically similar to our isolate, led us to hypothesize that the Euganean and PCC 8501 strains are either very similar sister species or ecotypes of the same species in a yet to be defined clade, clearly distinct within the paraphyletic Leptolyngbya group.