To obtain fast growing oil-rich microalgal strains has been urgently demanded for microalgal biofuel. Malic enzyme (ME), which is involved in pyruvate metabolism and carbon fixation, was first characterized in microalgae here. Overexpression of Phaeodactylum tricornutum ME (PtME) significantly enhanced the expression of PtME and its enzymatic activity in transgenic P. tricornutum. The total lipid content in transgenic cells markedly increased by 2.5-fold and reached a record 57.8% of dry cell weight with a similar growth rate to wild type, thus keeping a high biomass. The neutral lipid content was further increased by 31% under nitrogen-deprivation treatment, still 66% higher than that of wild type. Transgenic microalgae cells exhibited obvious morphological changes, as the cells were shorter and thicker and contained larger oil bodies. Immuno-electron microscopy targeted PtME to the mitochondrion. This study markedly increased the oil content in microalgae, suggesting a new route for developing ideal microalgal strains for industrial biodiesel production.

Cytosolic 10-kDa acyl-CoA-binding proteins (ACBPs) function in the storage and intracellular transport of acyl-CoA esters in eukaryotes. Fatty acids synthesized de novo in plant chloroplasts are exported as oleoyl-CoA and palmitoyl-CoA esters. In Arabidopsis, other than the 10-kDa ACBP, there exists five larger ACBPs (ACBP1 to ACBP5) of which homologues have not been characterized in other organisms. To investigate the significance of this gene family, we have attempted to subcellularly localize them and compare their acyl-CoA-binding affinities. We have previously shown that Arabidopsis ACBP1 and ACBP2 are membrane-associated proteins while ACBP4 and ACBP5 contain kelch motifs. Here, to localize ACBP3, we have expressed ACBP3-red fluorescent protein (DsRed2) from the CaMV 35S promoter. ACBP3-DsRed was localized extracellularly in transiently expressed tobacco BY-2 cells and onion epidermal cells. The function of the acyl-CoA-binding domain in ACBP3 was investigated by in vitro binding assays using (His)(6)-ACBP3, which was observed to bind [(14)C]arachidonyl-CoA with high affinity in comparison to [(14)C]palmitoyl-CoA and [(14)C]oleoyl-CoA. To identify the residues functional in binding, five mutants with single amino acid substitutions in the acyl-CoA-binding domain of (His)(6)-ACBP3 and (His)(6)-ACBP1 (which also binds [(14)C]arachidonyl-CoA) were generated by site-directed mutagenesis. Binding assays with arachidonyl-CoA revealed that replacement of a conserved R residue (R150A in ACBP1 and R284A in ACBP3), disrupted binding. In contrast, other substitutions in ACBP1 (Y126A, K130A, K152A and Y171A) and in ACBP3 (F260A, K264A, K286A and Y305A) did not affect arachidonyl-CoA binding, unlike their equivalents in (His)(6)-ACBP2, (His)(6)-ACBP4 and (His)(6)-ACBP5, which had altered binding to palmitoyl-CoA or oleoyl-CoA.

Genetic bottlenecks are stochastic events that limit genetic variation in a population and result in founding populations that can lead to genetic drift. Evidence of past genetic bottlenecks in numerous biological systems, from mammals to viruses, has been described. In this study, we used an artificial population of Cucumber mosaic virus consisting of 12 restriction enzyme marker-bearing mutants. This population was inoculated onto young leaves of tobacco plants and monitored throughout the course of systemic infection. We show here that the genetic variation in a defined population of an RNA virus is significantly, stochastically, and reproducibly reduced during the systemic infection process, providing clear evidence of a genetic bottleneck.Populations generate genetic variation over time, which can be specifically reduced by selection or stochastically reduced by genetic bottlenecks. After a bottleneck, a limited number of randomly selected individuals create a founding population, resulting in genetic drift. The occurrence of bottlenecks in nature has been inferred from the low genetic diversity of extant populations and extrapolation of an historical event (reviewed in reference 1).Genetic bottlenecks may occur frequently during the natural life cycles of RNA viruses. Transmission events, both horizontal and vertical, and systemic infections represent events in the virus life cycle that may impose a bottleneck. A few studies of plant viruses have attempted to estimate the effect of bottlenecks by examining diversity in populations from systemically infected tissues (18,40). In addition, a number of studies of animal and bacterial viruses have examined changes in the population structure after passage through artificial bottlenecks (4, 7, 32, 49), but naturally occurring bottlenecks have been poorly studied.RNA viruses are characterized by the potential for a high degree of variability due to short generation times and errorprone replication (reviewed in reference 11), leading to populations known as quasispecies (14,15). In spite of this potential, quasispecies variation in experimental evolution studies with plant viruses was significantly lower than what might be predicted (42).Low genetic diversity may result from low mutation rates. However, a rough estimation of the mutation rate of Tobacco mosaic virus suggested that plant viruses were not significantly different from animal RNA viruses (27). Selection (predominantly negative) for maintaining a functional encoded protein or functional RNA structure is believed to play an important role in the elimination of deleterious variants (20,34,36). Population bottlenecks during transmission might also be an important factor for limiting the variation in a population. This phenomenon was implicated in some laboratory passage experiments (2, 23, 25) as well as some analyses of natural plant virus populations (3, 6, 17). However, there hasn't been any direct experimental evidence showing that naturally occurring bottlenecks play a role in the genetic structure o...