The carbon concentrating mechanism in Chlamydomonas reinhardtii: finding the missing pieces.

The photosynthetic, unicellular green alga, Chlamydomonas reinhardtii, lives in environments that often contain low concentrations of CO2 and HCO3 (-), the utilizable forms of inorganic carbon (Ci). C. reinhardtii possesses a carbon concentrating mechanism (CCM) which can provide suitable amounts of Ci for growth and development. This CCM is induced when the CO2 concentration is at air levels or lower and is comprised of a set of proteins that allow the efficient uptake of Ci into the cell as well as its directed transport to the site where Rubisco fixes CO2 into biomolecules. While several components of the CCM have been identified in recent years, the picture is still far from complete. To further improve our knowledge of the CCM, we undertook a mutagenesis project where an antibiotic resistance cassette was randomly inserted into the C. reinhardtii genome resulting in the generation of 22,000 mutants. The mutant collection was screened using both a published PCR-based approach (Gonzalez-Ballester et al. 2011) and a phenotypic growth screen. The PCR-based screen did not rely on a colony having an altered growth phenotype and was used to identify colonies with disruptions in genes previously identified as being associated with the CCM-related gene. Eleven independent insertional mutations were identified in eight different genes showing the usefulness of this approach in generating mutations in CCM-related genes of interest as well as identifying new CCM components. Further improvements of this method are also discussed.

Photorespiration and carbon concentrating mechanisms: two adaptations to high O2, low CO2 conditions.

This review presents an overview of the two ways that cyanobacteria, algae, and plants have adapted to high O2 and low CO2 concentrations in the environment. First, the process of photorespiration enables photosynthetic organisms to recycle phosphoglycolate formed by the oxygenase reaction catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Second, there are a number of carbon concentrating mechanisms that increase the CO2 concentration around Rubisco which increases the carboxylase reaction enhancing CO2 fixation. This review also presents possibilities for the beneficial modification of these processes with the goal of improving future crop yields.

Members of a small family of nodulin-like genes are regulated under iron deficiency in roots of Arabidopsis thaliana.

The analysis of rapid responses in the transcriptome of Arabidopsis roots to a decreased iron (Fe) supply was studied using DNA microarrays and revealed candidate genes with putative roles in Fe homeostasis. In addition to the frequently reported induction of gene activity in response to Fe deficiency, the expression of a number of putative cationic metal transporters was found to rapidly decrease in response to Fe deficiency. In this report we have investigated a small family of five nodulin-like genes that show protein sequence similarity to AtVIT1 and likely have a function in regulation of Fe homeostasis. DNA microarray analysis showed a rapid decrease in transcript abundance for nodulin-like1 (At1g21140), nodulin-like2 (At1g76800), and nodulin-like21 (At3g25190). This decrease was significant after 6 h of Fe deficiency and persisted at least to 72 h. Nodulin-like3 (At3g43630) and Nodulin-like4 (At3g43660) did not respond to the Fe concentration in the microarray analysis. The nodulin-like family encoded presumptive membrane proteins with five calculated transmembrane domains, and all members had significant protein sequence homology to the vacuolar Fe transporters AtVIT1 and ScCCC1p. Homologs of all five nodulin-like genes were found in both di- and monocotyledon plants, as well as in Physcomitrella and Chlamydomonas. Promoter-ß-glucuronidase (GUS) assays showed expression of the nodulin-like1 gene in roots, hypocotyls, and expanded cotyledons of two-week-old Arabidopsis seedlings with the greatest activity associated with the vascular bundle and the root stele. In the absence of Fe, GUS activity was greatly reduced and was only weakly visible in the stele and vascular bundle. In an attempt to identify the function of these nodulin-like proteins, we isolated knockout mutants for nodulin-like3 and nodulin-like21 from available T-DNA insertion lines. Although these mutants did not show dramatic changes in growth or in their ability to grow on Fe-deficient media or media containing from 5 to 120 µM Fe, the nodulin-like3 mutant had a significantly higher Fe concentration in the shoots and both nodulin-like3 and nodulin-like21 mutants had significantly decreased Fe in the roots. These results were taken as an indication, that some members of this nodulin-like family were directly involved in Fe homeostasis in plants.

Hemagglutinin of influenza virus partitions into the nonraft domain of model membranes.

The HA of influenza virus is a paradigm for a transmembrane protein thought to be associated with membrane-rafts, liquid-ordered like nanodomains of the plasma membrane enriched in cholesterol, glycosphingolipids, and saturated phospholipids. Due to their submicron size in cells, rafts can not be visualized directly and raft-association of HA was hitherto analyzed by indirect methods. In this study, we have used GUVs and GPMVs, showing liquid disordered and liquid ordered domains, to directly visualize partition of HA by fluorescence microscopy. We show that HA is exclusively (GUVs) or predominantly (GPMVs) present in the liquid disordered domain, regardless of whether authentic HA or domains containing its raft targeting signals were reconstituted into model membranes. The preferential partition of HA into ld domains and the difference between lo partition in GUV and GPMV are discussed with respect to differences in packaging of lipids in membranes of model systems and living cells suggesting that physical properties of lipid domains in biological membranes are tightly regulated by protein-lipid interactions.