SUMMARY Developing renewable energy sources is critical to maintaining the economic growth of the planet while protecting the environment. First generation biofuels focused on food crops like corn and sugarcane for ethanol production, and soybean and palm for biodiesel production. Second generation biofuels based on cellulosic ethanol produced from terrestrial plants, has received extensive funding and recently pilot facilities have been commissioned, but to date output of fuels from these sources has fallen well short of what is needed. Recent research and pilot demonstrations have highlighted the potential of algae as one of the most promising sources of sustainable liquid transportation fuels. Algae have also been established as unique biofactories for industrial, therapeutic, and nutraceutical co-products. Chlamydomonas reinhardtii’s long established role in the field of basic research in green algae has paved the way for understanding algal metabolism and developing genetic engineering protocols. These tools are now being utilized in C. reinhardtii and in other algal species for the development of strains to maximize biofuels and bio-products yields from the lab to the field.

Background:The tomato leucine aminopeptidase (LAP) regulates wound signaling, and its mode of action is unknown. Results: Plant LAPs are molecular chaperones, and the chaperone activity of the tomato LAP-A is independent of its peptidase and enhanced upon hexamer disruption. Conclusion: Plant LAPs are bifunctional, with both aminopeptidase and chaperone activities. Significance: Plant LAPs are a new class of molecular chaperone with roles in plant defense.

Algae have enormous potential as bio-factories for the efficient production of a wide array of high-value products, and eventually as a source of renewable biofuels. However, tools for engineering the nuclear genomes of algae remain scarce and limited in functionality. In this study, synthetic algal promoters (saps) were generated as a tool for increasing nuclear gene expression and as a model for understanding promoter elements and structure in green algae. Promoters were generated to mimic native cis-motif elements, structure, and overall nucleotide composition of top expressing genes from Chlamydomonas reinhardtii. Twenty five saps were used to drive expression of a fluorescent report in transgenic algae. A majority of the promoters were functional in vivo and seven were identified to drive expression of the fluorescent reporter better than the current best endogenous promoter in C. reinhardtii, the chimeric hsp70/rbs2 promoter. Further analysis of the best synthetic promoter, sap11, revealed a new DNA motif essential for promoter function that is widespread and highly conserved in C. reinhardtii. These data demonstrate the utility of synthetic promoters to drive gene expression in green algae, and lays the groundwork for the development of a suite of saps capable of driving the robust and complex gene expression that will be required for algae to reach their potential as an industrial platform for photosynthetic biomanufacturing. 2008). This diversity has been exploited as a unique source of bioactive compounds, including antioxidants, omega 3 fatty acids, and potentially novel therapeutic drugs (Cardozo et al., 2007). In addition, microalgae have also proven to be cost-effective and safe hosts for expressing a wide array of recombinant proteins, including human and animal therapeutics, vaccines, and industrial

Oxygenic photosynthesis provides the energy to produce all food and most of the fuel on this planet. Photosystem II (PSII) is an essential and rate-limiting component of this process. Understanding and modifying PSII function could provide an opportunity for optimizing photosynthetic biomass production, particularly under specific environmental conditions. PSII is a complex multisubunit enzyme with strong interdependence among its components. In this work, we have deleted the six core genes of PSII in the eukaryotic alga Chlamydomonas reinhardtii and refactored them in a single DNA construct. Complementation of the knockout strain with the core PSII synthetic module from three different green algae resulted in reconstitution of photosynthetic activity to 85, 55, and 53% of that of the wild-type, demonstrating that the PSII core can be exchanged between algae species and retain function. The strains, synthetic cassettes, and refactoring strategy developed for this study demonstrate the potential of synthetic biology approaches for tailoring oxygenic photosynthesis and provide a powerful tool for unraveling PSII structure-function relationships.

Wounding due to mechanical injury or insect feeding causes a wide array of damage to plant cells including cell disruption, desiccation, metabolite oxidation, and disruption of primary metabolism. In response, plants regulate a variety of genes and metabolic pathways to cope with injury. Tomato (Solanum lycopersicum) is a model for wound signaling but few studies have examined the comprehensive gene expression profiles in response to injury. A cross-species microarray approach using the TIGR potato 10-K cDNA array was analyzed for large-scale temporal (early and late) and spatial (locally and systemically) responses to mechanical wounding in tomato leaves. These analyses demonstrated that tomato regulates many primary and secondary metabolic pathways and this regulation is dependent on both timing and location. To determine if LAP-A, a known modulator of wound signaling, influences gene expression beyond the core of late wound-response genes, changes in RNAs from healthy and wounded Leucine aminopeptidase A-silenced (LapA-SI) and wild-type (WT) leaves were examined. While most of the changes in gene expression after wounding in LapA-SI leaves were similar to WT, overall responses were delayed in the LapA-SI leaves. Moreover, two pathogenesis-related 1 (PR-1c and PR-1a2) and two dehydrin (TAS14 and Dhn3) genes were negatively regulated by LAP-A. Collectively, this study has shown that tomato wound responses are complex and that LAP-A’s role in modulation of wound responses extends beyond the well described late-wound gene core.