Bacterial luciferase as a reporter of circadian gene expression in cyanobacteria.

To allow continuous monitoring of the circadian clock in cyanobacteria, we previously created a reporter strain (AMC149) of Synechococcus sp. strain PCC 7942 in which the promoter of the psbAI gene was fused to Vibrio harveyi luciferase structural genes (luxAB) and integrated into the chromosome. Northern (RNA) hybridization and immunoblot analyses were performed to examine changes in abundance of the luxAB mRNA, the native psbAI mRNA, and the luciferase protein to determine whether bioluminescence is an accurate reporter of psbAI promoter activity in AMC149. Under constant light conditions, the mRNA abundances of both luxAB and psbAI oscillated with a period of approximately 24 h for at least 2 days. The expression of these two genes following the same pattern: both mRNAs peaked in the subjective morning, and their troughs occurred near the end of the subjective night. The amount of luciferase protein also oscillated with a period of approximately 24 h, and the protein rhythm is in phase with the bioluminescence rhythm. The rhythm of the luciferase mRNA phase-leads the rhythms of luciferase protein and in vivo bioluminescence by several hours. Comparable results were obtained with a short-period mutant of AMC149. Together, these results indicate that the bioluminescence rhythm in AMC149 is due primarily to circadian oscillation of psbAI promoter activity in this cyanobacterium.     

Light availability influences the ratio of two forms of D1 in cyanobacterial thylakoids

The psbA multigene family in Synechococcus sp. strain PCC 7942 encodes two forms of the D1 protein; Form I, the product of psbAI, differs from Form II, the product of psbAII and psbAIII, at 25 of 360 amino acid positions. D1 is essential for photosynthesis as a protein component of the photosystem II reaction center. Antisera were raised against purified hybrid proteins encoded by psbAI-lacZ and psbAIII-lacZ translational gene fusions that contain the unique amino termini of Form I and Form II, respectively. Form specificity of each antiserum was verified by Western analysis using thylakoid membranes from mutant strains containing only Form I or Form II. Western analysis of thylakoid membranes from wild-type cells cultured at different light intensities detected both forms of D1 in the membrane and showed changes in the ratio of the two forms. The D1 composition of the membrane matched predicted ratios of the forms based on differential gene expression: psbAI is expressed highest at low light, and both psbAII and psbAIII are expressed highest at high light. Along a gradient of light intensity from 5 microE. m-2.s-1 to 482 microE.m-2.s-1, the relative amount of Form I in thylakoid membranes decreased 58%, while the relative amount of Form II increased 60%. Maximum detection of Form I coupled with minimum detection of Form II in membranes from cells harvested at light intensities below 390 microE.m-2.s-1 suggests a central role for Form I in photosystem II. Increased incorporation of Form II into the thylakoid membrane occurred at light intensities reported by others to be photoinhibitory, suggesting that Form II serves a role in adaptation to high light.     

Thermoluminescence and flash-induced oxygen yield in herbicide resistant mutants of the D1 protein in Synechococcus PCC7942.

Several strains of Synechococcus PCC7942 carrying point mutations in the gene psbA were studied by thermoluminescence and polarographic measurement of flash-induced oxygen yield. The following results were obtained: (a) Replacement of Ser-264 in D1 by Ala (mutant Di1) or Gly (mutant G264) resulting in DCMU and atrazine resistance leads to a downshift of the thermoluminescence (TL) B-band peak temperature from 40 degrees C in wild-type thylakoids to about 30 degrees C. In dark adapted samples of both mutants the TL and oxygen yield pattern induced by a train of single turnover flashes were strongly damped indicative of a high miss factor. (b) In contrast to Ser-264 mutants, replacement of Phe-255 in D1 by Tyr (mutant Tyr5) induced strong resistance to atrazine but not to DCMU and did not affect the peak termperature of the B-band and the flash-induced TL and oxygen yield patterns. In this respect mutant Tyr5 resembles the wild type. (c) No significant differences have been found between strains with single site mutations in psbAI and normal psbAII/psbAIII genes, and strains with same mutations in psbAI but additional deletion of psbAII and psbAIII. Obviously in strains were psbAI is present, PS II complexes containing gene products of psbAII and psbAIII are not assembled in detectable amounts. (d) Strains with double mutations at positions 264 and 255 display a downshift of the B-band peak temperature. Their oscillatory patterns of B-band intensity and oxygen yield are highly damped. This behaviour is similar to strains D1 and G264 which are modified at position 264 only. We extend reports on additivity of mutation effects on herbicide binding to binding of QB. (e) Mutations at the QB site not only influence the binding of QB and herbicides but also change the thermoluminescence quantum yield and the lifetimes of the redox states S2 and S3 of the water oxidase. This finding might indicate long ranging effects on Photosystem II exerted by structural modifications of the QB site. From these data we conclude that Ser-264 is essential for binding of atrazine, DCMU and QB, whereas Phe-255 is involved in atrazine binding and its substitution by Tyr does not markedly affect QB or DCMU binding in Synechococcus PCC7942.     

Mutation in phenol-type herbicide resistance maps within the psbA gene in Synechocystis 6714

A Synechocytis 6714 mutant resistant to the phenol-type herbicide ioxynil was isolated and characterized. Sensitivity to DCMU and atrazine was tf measured in whole cells and isolated thylakoids. The mutant presents the same sensitivity to atrazine as the wild type and a slightly increased sensitivity to DCMU. A point mutation has been found at codon 266 in the psbAI coding locus (AAC to ACC) resulting in an amino acid change from asparagine to threonine in the D1 protein.     

Electron transport regulates exchange of two forms of photosystem II D1 protein in the cyanobacterium Synechococcus.

Synechococcus sp. PCC 7942 modulates photosynthetic function by transiently replacing the constitutive D1 photosystem II protein, D1:1, with an alternate form, D1:2, to help counteract photoinhibition under excess light. We show that a temperature drop from 37 to 25 degrees C also drives D1:1/D1:2 exchange under constant, moderate light. Chilling or light-induced D1 exchange results from rapid loss of psbAI message coding for D1:1 and accumulation of psbAII and psbAIII messages coding for D1:2. During chilling, a large pool of a novel form, D1:2*, transiently accumulates, distinguishable from normal D1 by an increase in apparent molecular mass. D1:2* is not phosphorylated and is probably a functionally inactive, incompletely processed precursor. After acclimation to 25 degrees C, D1:2* disappears and D1:1 again predominates, although substantial D1:2 remains. Partial inhibition of electron transport under constant, moderate light also triggers the D1 exchange process. These treatments all increase excitation pressure on photosystem II relative to electron transport. Therefore, information from photosynthetic electron transport regulates D1 exchange without any requirement for a change in light intensity or quality, possibly via a redox sensing mechanism proximal to photosystem II.     

Quality control of photosystem II

Photosystem II is particularly vulnerable to excess light. When illuminated with strong visible light, the reaction center D1 protein is damaged by reactive oxygen molecules or by endogenous cationic radicals generated by photochemical reactions, which is followed by proteolytic degradation of the damaged D1 protein. Homologs of prokaryotic proteases, such as ClpP, FtsH and DegP, have been identified in chloroplasts, and participation of the thylakoid-bound FtsH in the secondary degradation steps of the photodamaged D1 protein has been suggested. We found that cross-linking of the D1 protein with the D2 protein, the alpha-subunit of cytochrome b(559), and the antenna chlorophyll-binding protein CP43, occurs in parallel with the degradation of the D1 protein during the illumination of intact chloroplasts, thylakoids and photosystem II-enriched membranes. The cross-linked products are then digested by a stromal protease(s). These results indicate that the degradation of the photodamaged D1 protein proceeds through membrane-bound proteases and stromal proteases. Moreover, a 33-kDa subunit of oxygen-evolving complex (OEC), bound to the lumen side of photosystem II, regulates the formation of the cross-linked products of the D1 protein in donor-side photoinhibition of photosystem II. Thus, various proteases and protein components in different compartments in chloroplasts are implicated in the efficient turnover of the D1 protein, thus contributing to the control of the quality of photosystem II under light stress conditions.