Circadian gating of the psbAIII high light response in Synechococcus sp. strain PCC 7942

Abstract

Synechococcus sp. strain PCC 7942 is a unicellular cyanobacterium which performs oxygenic photosynthesis by means of two photosystems analogous to higher plants (4). The photosystem II reaction center core contains a dimer composed of proteins D1 and D2. D1 is encoded by a three member psbA family: psbAI, psbAII, and psbAIII which are differentially regulated in response to changes in light intensity (10, 11). When cells are shifted from low light (100 []Em⁻²*s⁻¹) to high light (>500 []Em⁻²*s⁻¹), the psbAII and psbAIII transcript levels increase rapidly, while the psbAI level decreases (7). Recently, an additional level of light response regulation was identified. PCC 7942 possesses a circadian oscillator that appears to gate the psbAIII light response such that the response is "allowed" only at certain times during the circadian cycle. Because all three psbA transcripts have been demonstrated to cycle in a circadian manner, we used a PpsbAIII::luxAB (luciferase) transcriptional fusion to monitor by bioluminescence both the circadian rhythm and the light response. In cultures maintained at constant cell density we found the response to the high light shift of the PpsbAIII::luxAB to be highest (15-40 fold) during the troughs of the circadian cycle and lowest (1-5 fold) during the peaks of the cycle. We also found that in a clock null strain the lack of an oscillator does not entirely negate the light response of PpsbAIII::luxAB; however, this response does not demonstrate gating. In contrast to the bioluminescence data, northern analysis utilizing a psbAIII specific RNA probe exhibited the lack of circadian gating for the message in wild type cells. Additionally, little difference in D1 Form II between a clock wild type strain and a clock null strain was detected and there was no observable gating within sensitivity limits of western analysis. Finally, β-galactosidase assays were also in contradiction with the bioluminescence data. Assays performed in strains with a wild type clock background failed to exhibit a gated psbAIII high light response. This body of work conveys the rationale that while the luciferase reporter is an amiable method to monitor gene expression, under certain conditions the bioluminescence data must be carefully considered.