Abstract
A regulatory gene, designated pfsR (photosynthesis, Fe homeostasis and stress-response regulator), was discovered by a genetic screen in Synechocystis PCC 6803. Deletion of the gene from a high light-sensitive strain lacking four hli genes (4Xhli) restored viability to the parental strain under high light conditions. The quintuple mutant pfsR-/4Xhli retained photosystem-II and oxygen evolution capacity at levels similar to the wild-type levels under high light conditions. The transcripts of the two bfr genes (encoding bacterioferritin) were found to be constitutively up-regulated, whereas the transcripts of ho1 gene (encoding a heme oxygenase) were greatly down-regulated in high light upon deletion of pfsR. Under intermediate high intensity light, the pfsR deletion strains accumulated carotenoids and chlorophyll a to a significantly higher level than their corresponding parental strains. An exacerbated, transient increase in oxygen evolution during the early hours of high light acclimation and a somewhat increased steady-state level of photosystem-II-mediated oxygen evolution observed in the 4Xhli strain were brought back to the wild-type levels upon deletion of pfsR from the strain. The pfsR deletion mutants were found to be less sensitive to iron limitation under low light conditions and to suffer less lipid peroxidation following exposure to high light. Therefore, inactivation of PfsR resulted in tighter control of iron availability, which in turn reduced oxidative stress during photosynthesis in high light. These studies have revealed a critical role of PfsR in regulation of iron homeostasis and stress response.
Highlights
Both microalgae and vascular plants have evolved mechanisms for photo-acclimation that enable them to tolerate the absorption of excess excitation energy and limited amounts of reactive oxygen [3,4,5,6]
The genetic screen has resulted in the discovery of the regulatory gene pfsR, corresponding to the open reading frame sll1392 in Synechocystis PCC 6803
Identification of the Suppressor Mutation in the Suppressor pfs1—To identify the suppressor mutation in pfs1, the 4Xhli mutant was transformed with a recombinant genomic library of pfs1, and transformants were screened for complementation of the defect in high light (HL) tolerance as described under “Experimental Procedures.”
Summary
Culture Growth Conditions and Light and Iron Treatments— Synechocystis PCC 6803 strain was cultivated in BG-11 medium with 10 mM TES, pH 8.2, at 30 °C. A total of six stable suppressors, named “pfs1– 6” (the naming reflects our wishes to isolate mutants involve in photosynthesis, Fe homeostasis and stress responses), were obtained These suppressors grow well in LL with growth rates that are not significantly different from that of the wild type. The genomic regions flanking the inserted plasmid DNA, which delimits the putative complementing fragments, were determined by direct sequencing using the universal forward and reverse primers that bind at the multiple cloning site of pGEM-T, as previously reported [38]. Transformants were restreaked onto the same medium and segregation of the inactivated pfsR gene was monitored by PCR using genomic DNA of transformants and the two inner primers 5Ј-CCAAACGAAAGTTCAACCCCTAGA3Ј and 5Ј-ATAGAGGCTCCTGATAGCGGTTTTT-3Ј that recognize sequences upstream or downstream of the inserted eryr cassette. The numerical data are expressed as means Ϯ S.D
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