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. © 2014 Springer Science+Business Media Dordrecht

Plastid-Targeted Azelaic Acid-Induced 1 Family Proteins Mediate Systemic Defense Priming and Root Development during Arabidopsis Stress Signaling

The lipid transfer protein AZI1 is required for the priming of various systemic defense responses including: SAR (systemic acquired resistance); ISR (induced systemic resistance); and the uptake and mobilization of azelaic acid (AZA). Generated from the oxidation of plastid lipids during infections, AZA primes systemic defenses when applied to leaves or roots and induces striking changes in root development. These changes require the development- and defense-associated kinase MPK3, which is also required for SAR, ISR, and sensitivity to AZA. The AZI1 family also influences root development during salt stress and in response to AZA treatment similar to MPK3, which suggests the AZI1 protein family may serve as downstream coordinators of MPK3-dependent development and stress responses. Furthermore, MPK3 promotes the accumulation of AZI1 at plastids during infections, which suggests a critical role for AZI1’s plastid association in defense signaling. AZI1 is highly dynamic and traffics along the contact sites among the plastid, ER, and plasma membranes. Unlike canonical “signal anchored” proteins that target plastid membranes, AZI1’s normal pattern of plastid association requires a bipartite targeting signal that consists of a N-terminal hydrophobic domain and an internal proline-rich region. Including AZI1, the AZI1 gene family consists of seven short genes tandemly aligned on chromosome 4. Except for AZI5/AZI6 and AZI7, which have a shortened or absent PRR, respectively, AZI1 family proteins show a similar localization pattern as AZI1 and contribute to the priming of systemic immunity

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. © 2014 Springer Science+Business Media Dordrecht