Chromist algae (stramenopiles, cryptophytes, and haptophytes) are major contributors to marine primary productivity. These eukaryotes acquired their plastid via secondary endosymbiosis, whereby an early-diverging red alga was engulfed by a protist and the plastid was retained and its associated nuclear-encoded genes were transferred to the host genome. Current data suggest, however, that chromists are paraphyletic; therefore, it remains unclear whether their plastids trace back to a single secondary endosymbiosis or, alternatively, this organelle has resulted from multiple independent events in the different chromist lineages. Both scenarios, however, predict that plastid-targeted, nucleus-encoded chromist proteins should be most closely related to their red algal homologs. Here we analyzed the biosynthetic pathway of carotenoids that are essential components of all photosynthetic eukaryotes and find a mosaic evolutionary origin of these enzymes in chromists. Surprisingly, about one-third (5/16) of the proteins are most closely related to green algal homologs with three branching within or sister to the early-diverging Prasinophyceae. This phylogenetic association is corroborated by shared diagnostic indels and the syntenic arrangement of a specific gene pair involved in the photoprotective xanthophyll cycle. The combined data suggest that the prasinophyte genes may have been acquired before the ancient split of stramenopiles, haptophytes, cryptophytes, and putatively also dinoflagellates. The latter point is supported by the observed monophyly of alveolates and stramenopiles in most molecular trees. One possible explanation for our results is that the green genes are remnants of a cryptic endosymbiosis that occurred early in chromalveolate evolution; that is, prior to the postulated split of stramenopiles, alveolates, haptophytes, and cryptophytes. The subsequent red algal capture would have led to the loss or replacement of most green genes via intracellular gene transfer from the new endosymbiont. We argue that the prasinophyte genes were retained because they enhance photosynthetic performance in chromalveolates, thus extending the niches available to these organisms. The alternate explanation of green gene origin via serial endosymbiotic or horizontal gene transfers is also plausible, but the latter would require the independent origins of the same five genes in some or all the different chromalveolate lineages.

In vivo the prasinophyceaen alga Mantoniella squamata Manton et Parke uses an incomplete violaxanthin (Vx) cycle, leading to a strong accumulation of antheraxanthin (Ax) under conditions of high light. Here, we show that this zeaxanthin (Zx)-depleted Vx/Ax cycle is caused by an extremely slow second de-epoxidation step from Ax to Zx, and a fast epoxidation from Ax back to Vx in the light. The rate constant of Ax epoxidation is 5 to 6 times higher than the rate constant of Zx formation, implying that Ax is efficiently converted back to Vx before it can be de-epoxidated to Zx. It is, however, only half the rate constant of the first de-epoxidation step from Vx to Ax, thus explaining the observed net accumulation of Ax during periods of strong illumination. When comparing the rate constant of the second de-epoxidation step in M. squamata with Zx formation in spinach (Spinacia oleracea L.) thylakoids, we find a 20-fold reduction in the reaction kinetics of the former. This extremely slow Ax de-epoxidation, which is also exhibited by the isolated Mantoniella violaxanthin de-epoxidase (VDE), is due to a reduced substrate affinity of M. squamata VDE for Ax compared with the VDE of higher plants. Mantoniella VDE, which has a similar Km value for Vx, shows a substantially increased Km for the substrate Ax in comparison with spinach VDE. Our results furthermore explain why Zx formation in Mantoniella cells can only be found at low pH values that represent the pH optimum of VDE. A pH of 5 blocks the epoxidation reaction and, consequently, leads to a slow but appreciable accumulation of Zx.

The Sleeping Beauty (SB) transposon system can mediate stable gene transfer and expression in primary human T cells. Optimal in vitro conditions for maximum gene transfer efficiencies have been developed with regard to further application of the SB transposon system in T cell based gene therapies. This raises the question of whether or not the SB transposon system is a convincing alternative for virus-mediated gene transfer based on the currently available data. Here, we will discuss controversial safety and efficacy issues in transposon and viral gene transfer technology.