Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the~120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.

Comparing photosynthetic and photovoltaic efficiencies is not a simple issue. Although both processes harvest the energy in sunlight, they operate in distinctly different ways and produce different types of products: biomass or chemical fuels in the case of natural photosynthesis and nonstored electrical current in the case of photovoltaics. In order to find common ground for evaluating energy-conversion efficiency, we compare natural photosynthesis with present technologies for photovoltaic-driven electrolysis of water to produce hydrogen. Photovoltaic-driven electrolysis is the more efficient process when measured on an annual basis, yet short-term yields for photosynthetic conversion under optimal conditions come within a factor of 2 or 3 of the photovoltaic benchmark. We consider opportunities in which the frontiers of synthetic biology might be used to enhance natural photosynthesis for improved solar energy conversion efficiency.

Pro has been shown to play an important role in ameliorating environmental stress in plants and microorganisms, including heavy metal stress. Here, we describe the effects of the expression of a mothbean delta(1)-pyrroline-5-carboxylate synthetase (P5CS) gene in the green microalga Chlamydomonas reinhardtii. We show that transgenic algae expressing the mothbean P5CS gene have 80% higher free-Pro levels than wild-type cells, grow more rapidly in toxic Cd concentrations (100 microM), and bind fourfold more Cd than wild-type cells. In addition, Cd-K edge extended x-ray absorption fine structure studies indicated that Cd does not bind to free Pro in transgenic algae with increased Pro levels but is coordinated tetrahedrally by sulfur of phytochelatin. In contrast to P5CS-expressing cells, Cd is coordinated tetrahedrally by two oxygen and two sulfur atoms in wild-type cells. Measurements of reduced/oxidized GSH ratios and analyses of levels of malondialdehyde, a product of the free radical damage of lipids, indicate that free Pro levels are correlated with the GSH redox state and malondialdehyde levels in heavy metal-treated algae. These results suggest that the free Pro likely acts as an antioxidant in Cd-stressed cells. The resulting increased GSH levels facilitate increased phytochelatin synthesis and sequestration of Cd, because GSH-heavy metal adducts are the substrates for phytochelatin synthase.

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More than 250 million Africans rely on the starchy root crop cassava (Manihot esculenta) as their staple source of calories. A typical cassava-based diet, however, provides less than 30% of the minimum daily requirement for protein and only 10%-20% of that for iron, zinc, and vitamin A. The BioCassava Plus (BC+) program has employed modern biotechnologies intended to improve the health of Africans through the development and delivery of genetically engineered cassava with increased nutrient (zinc, iron, protein, and vitamin A) levels. Additional traits addressed by BioCassava Plus include increased shelf life, reductions in toxic cyanogenic glycosides to safe levels, and resistance to viral disease. The program also provides incentives for the adoption of biofortified cassava. Proof of concept was achieved for each of the target traits. Results from field trials in Puerto Rico, the first confined field trials in Nigeria to use genetically engineered organisms, and ex ante impact analyses support the efficacy of using transgenic strategies for the biofortification of cassava.

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One of the major constraints facing the large-scale production of cassava (Manihot esculenta) roots is the rapid postharvest physiological deterioration (PPD) that occurs within 72 h following harvest. One of the earliest recognized biochemical events during the initiation of PPD is a rapid burst of reactive oxygen species (ROS) accumulation. We have investigated the source of this oxidative burst to identify possible strategies to limit its extent and to extend cassava root shelf life. We provide evidence for a causal link between cyanogenesis and the onset of the oxidative burst that triggers PPD. By measuring ROS accumulation in transgenic low-cyanogen plants with and without cyanide complementation, we show that PPD is cyanide dependent, presumably resulting from a cyanide-dependent inhibition of respiration. To reduce cyanide-dependent ROS production in cassava root mitochondria, we generated transgenic plants expressing a codon-optimized Arabidopsis (Arabidopsis thaliana) mitochondrial alternative oxidase gene (AOX1A). Unlike cytochrome c oxidase, AOX is cyanide insensitive. Transgenic plants overexpressing AOX exhibited over a 10-fold reduction in ROS accumulation compared with wild-type plants. The reduction in ROS accumulation was associated with a delayed onset of PPD by 14 to 21 d after harvest of greenhouse-grown plants. The delay in PPD in transgenic plants was also observed under field conditions, but with a root biomass yield loss in the highest AOX-expressing lines. These data reveal a mechanism for PPD in cassava based on cyanideinduced oxidative stress as well as PPD control strategies involving inhibition of ROS production or its sequestration.

Cassava ( Manihot esculenta Crantz.) is the major source of calories for subsistence farmers in sub-Saharan Africa. Cassava, however, contains potentially toxic levels of the cyanogenic glucoside, linamarin. The cyanogen content of cassava foods can be reduced to safe levels by maceration, soaking, rinsing and baking; however, short-cut processing techniques can yield toxic food products. Our objective was to eliminate cyanogens from cassava so as to eliminate the need for food processing. To achieve this goal we generated transgenic acyanogenic cassava plants in which the expression of the cytochrome P450 genes ( CYP79D1 and CYP79D2), that catalyze the first-dedicated step in linamarin synthesis, was inhibited. Using a leaf-specific promoter to drive the antisense expression of the CYP79D1/ CYP79D2 genes we observed up to a 94% reduction in leaf linamarin content associated with an inhibition of CYP79D1 and CYP79D2 expression. Importantly, the linamarin content of roots also was reduced by 99% in transgenic plants having between 60 and 94% reduction in leaf linamarin content. Analysis of CYP79D1/ CYP79D2 transcript levels in transgenic roots indicated they were unchanged relative to wild-type plants. These results suggest that linamarin is transported from leaves to roots and that a threshold level of leaf linamarin production is required for transport.