Especially when excluding any influence of PSII photochemistry by

Especially when excluding any influence of PSII photochemistry by adding

DCMU, the changes of the PSII antennae size upon state transition can be directly followed by changes of chlorophyll fluorescence yields (Finazzi et al. 2001a, b). These changes in fluorescence can be visualized by the abovementioned video imaging system, which has been described in detail, e.g., by Fenton and Roscovitine Crofts (1990) and by Kruse et al. (1999). This system significantly simplifies the whole screening procedure of even large Chlamydomonas GS-9973 price transformant libraries. The generation of the latter usually begins with transformation of the cells by a selectable marker gene. The transformed cells are then plated on selective agar plates. On these first plates, successfully transformed clones grow in unorganized patterns. Most screening procedures require the transfer of every single colony to new master plates in an organized raster, so that several thousand clones have to be transferred, though only a tiny fraction of them will turn out to have the desired phenotype. In contrast, the fluorescence imaging system allows screening the algal colonies already on the first, unorganized agar plates, given that the colonies have approximately the same size, which usually is the case. Furthermore, the strategies used in order to force C. reinhardtii cells into state 1 or state

2 are applicable on whole agar plates. Fleischmann et al. (1999) plated the transformed cells directly on TAP agar plates containing

DCMU and incubated the plates in low MK0683 light (6 μE m−2 s−1). As mentioned above, the inhibition of PSII photochemistry allows to directly concluding the state from PSII fluorescence at room temperature. In these DCMU-treated algal colonies, state 1 could then easily be achieved cAMP by illuminating the cells with white light, resulting in the oxidation of the PQ pool by PSI activity. State 2 was achieved by making use of the fact that anaerobic and dark-incubated C. reinhardtii cells have a reduced PQ pool and therefore shift to state 2 (Wollman and Delepelaire 1984). With an appropriate setup, whole Petri dishes can be flushed with N2 in the dark, forcing the algal colonies into state 2 (Fleischmann et al. 1999). Applying these treatments to the agar plates harboring Chlamydomonas transformant colonies, fluorescence pictures of the whole plates can be recorded and numerically subtracted, so that the fluorescence difference of each colony provides a measure of state transition. While C. reinhardtii wild-type colonies display strong signals, strains deficient in state transitions show weak or nearly undetectable signals (Fleischmann et al. 1999). Kruse et al. (1999) used a similar technical setup, but applied a different strategy to induce state transitions in the microalgae.

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