Current ambient UV-B levels can significantly depress productivity in aquatic habitats, largely because UV-B inhibits several steps of photosynthesis, including the photooxidation of water catalyzed by photosystem II. We show that upon UV-B exposure the cyanobacterium Synechococcus sp. PCC 7942 rapidly changes the expression of a family of three psbA genes encoding photosystem II D1 proteins. In wild-type cells the psbAI gene is expressed constitutively, but strong accumulations of psbAII and psbAIII transcripts are In oxygenic photobionts, photosystem II (PSII) is an integral membrane complex that catalyzes the photooxidation of water, with concomitant release of oxygen. Electrons extracted from water are passed to plastoquinone and enter the photosynthetic electron transport chain. The core of the PSII complex is composed of a dimer of two related proteins, D1 and D2, that bind the pigments and cofactors involved in this electron transfer from water to plastoquinone. During active photosynthesis the D1 protein, and to a lesser extent D2, turn over rapidly and are replaced by newly synthesized polypeptides in a PSII repair cycle. Under environmental stress, the repair cycle can be impaired, such that degradation and loss of D1 protein exceeds the rate of replacement (1). This net loss of functional D1 leads to a drop in PSII function and can contribute to photoinhibition, a light-dependent drop in the quantum yield of photosynthesis. Photoinhibition usually occurs when excitation capture exceeds the rate of electron removal from the PSII complex, as can occur when the light intensity exceeds the acclimated irradiance or when the temperature drops below the acclimated level.

The heat shock protein ClpB (HSP100) is a member of the diverse group of Clp polypeptides that function as molecular chaperones and/or regulators of energy-dependent proteolysis. A single-copy gene coding for a ClpB homolog was cloned and sequenced from the unicellular cyanobacterium Synechococcus sp. strain PCC 7942. The predicted polypeptide sequence was most similar to sequences of cytosolic ClpB from bacteria and higher plants (i.e., 70 to 75%). Inactivation of clpB in Synechococcus sp. strain PCC 7942 resulted in no significant differences from the wild-type phenotype under optimal growth conditions. In the wild type, two forms of ClpB were induced during temperature shifts from 37 to 47.5 or 50؇C, one of 92 kDa, which matched the predicted size, and another smaller protein of 78 kDa. Both proteins were absent in the ⌬clpB strain. The level of induction of the two ClpB forms in the wild type increased with increasingly higher temperatures, while the level of the constitutive ClpC protein remained unchanged. In the ⌬clpB strain, however, the ClpC content almost doubled during the heating period, presumably to compensate for the loss of ClpB activity. Photosynthetic measurements at 47.5 and 50؇C showed that the null mutant was no more susceptible to thermal inactivation than the wild type. Using photosynthesis as a metabolic indicator, an assay was developed for Synechococcus spp. to determine the importance of ClpB for acquired thermotolerance. Complete inactivation of photosynthetic oxygen evolution occurred in both the wild type and the ⌬clpB strain when they were shifted from 37 directly to 55؇C for 10 min. By preexposing the cells at 50؇C for 1.5 h, however, a significant level of photosynthesis was retained in the wild type but not in the mutant after the treatment at 55؇C for 10 min. Cell survival determinations confirmed that the loss of ClpB synthesis caused a fivefold reduction in the ability of Synechococcus cells to develop thermotolerance. These results clearly show that induction of ClpB at high temperatures is vital for sustained thermotolerance in Synechococcus spp., the first such example for either a photosynthetic or a prokaryotic organism.

The presence of genes encoding organellar proteins in different cellular compartments necessitates a tight coordination of expression by the different genomes of the eukaryotic cell. This coordination of gene expression is achieved by organelle-to-nucleus communication. Stress-induced perturbations of the tetrapyrrole pathway trigger large changes in nuclear gene expression. In order to investigate whether the tetrapyrrole Mg-ProtoIX itself is an important part of plastid-to-nucleus communication, we used an affinity column containing Mg-ProtoIX covalently linked to an Affi-Gel matrix. The proteins that bound to Mg-ProtoIX were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis combined with nano liquid chromatography-mass spectrometry (MS)/MS. Thus, we present a novel proteomic approach to address the mechanisms involved in cellular signaling and we identified interactions between Mg-ProtoIX and a large number of proteins associated with oxidative stress responses. Our approach revealed an interaction between Mg-ProtoIX and the heat shock protein 90-type protein, HSP81-2 suggesting that a regulatory complex including HSP90 proteins and tetrapyrroles controlling gene expression is evolutionarily conserved between yeast and plants. In addition, our list of putative Mg-ProtoIX-binding proteins demonstrated that binding of tetrapyrroles does not depend on a specific amino acid motif but possibly on a specific fold of the protein.

ClpB is a highly conserved heat shock protein that is essential for thermotolerance in bacteria and eukaryotes. One distinctive feature of all bacterial clpB genes is the dual translation of a truncated 79-kDa form (ClpB-79) in addition to the full-length 93-kDa protein (ClpB-93). To investigate the currently unknown function of ClpB-79, we have examined the ability of the two different-sized ClpB homologues from the cyanobacterium Synechococcus sp. strain PCC 7942 to confer thermotolerance. We show that the ClpB-79 form has the same capacity as ClpB-93 to confer thermotolerance and that the ClpB-79 protein contributes ca. one-third of the total thermotolerance developed in wild-type Synechococcus, the first in vivo demonstration of a functional role for ClpB-79 in bacteria.Changing environmental conditions elicit the synthesis of new types of cellular proteins in all organisms. This conserved molecular response is best exemplified by high-temperature stress, during which specific groups of polypeptides known as heat shock proteins (HSPs) are rapidly induced. Many of these HSPs are now known to function as molecular chaperones and are part of larger protein families that include constitutive members. Protein denaturation and aggregation are the major types of cellular damage that result from high temperatures, and HSP chaperones respond by preventing aggregation, assisting refolding, and targeting misfolded protein for degradation (13). The activity of such chaperones is essential for cell survival during heat shock and for subsequent recovery.ClpB is a heat shock-inducible representative of the HSP100/Clp family of oligomeric ATPases. The family can be divided into several types based on specific sequence signatures and other structural characteristics (17). Most member proteins are large (79 to 105 kDa) and contain two distinct nucleotide-binding domains separated by a spacer region of variable length (ClpA-E), whereas some are smaller (50 kDa) and have only one such domain (ClpX and ClpY). The various HSP100/Clp proteins are thought to function as molecular chaperones, with a common mechanism of dismantling multimeric protein complexes, as shown for ClpA and -X in Escherichia coli (7,22).ClpB is an HSP found in almost all organisms studied to date. Separate nuclear genes encode two different-sized ClpB proteins in yeast, a 78-kDa protein localized in mitochondria (6) and a 104-kDa protein localized primarily in the cytosol (16). Both ClpB proteins function as molecular chaperones. Along with DnaK (HSP70), mitochondrial ClpB helps prevent protein denaturation and aggregation at high temperatures (18, 19), whereas cytosolic ClpB (HSP104) disassembles large protein aggregates that accumulate at extreme high temperatures (14). Cytosolic ClpB also acts in concert with DnaK or DnaJ to promote the refolding of the once aggregated polypeptides (4). Such functions are thought to underlie the necessity of cytosolic ClpB for the acquisition of thermotolerance in yeast (16), which is the tolerance developed to a normally...