Comparative Analysis of Expressed Sequence Tag (EST) Libraries in the Seagrass Zostera marina Subjected to Temperature Stress

Global warming is associated with increasing stress and mortality on temperate seagrass beds, in particular during periods of high sea surface temperatures during summer months, adding to existing anthropogenic impacts, such as eutrophication and habitat destruction. We compare several expressed sequence tag (EST) in the ecologically important seagrass Zostera marina (eelgrass) to elucidate the molecular genetic basis of adaptation to environmental extremes. We compared the tentative unigene (TUG) frequencies of libraries derived from leaf and meristematic tissue from a control situation with two experimentally imposed temperature stress conditions and found that TUG composition is markedly different among these conditions (all P < 0.0001). Under heat stress, we find that 63 TUGs are differentially expressed (d.e.) at 25°C compared with lower, no-stress condition temperatures (4°C and 17°C). Approximately one-third of d.e. eelgrass genes were characteristic for the stress response of the terrestrial plant model Arabidopsis thaliana. The changes in gene expression suggest complex photosynthetic adjustments among light-harvesting complexes, reaction center subunits of photosystem I and II, and components of the dark reaction. Heat shock encoding proteins and reactive oxygen scavengers also were identified, but their overall frequency was too low to perform statistical tests. In all conditions, the most abundant transcript (3–15%) was a putative metallothionein gene with unknown function. We also find evidence that heat stress may translate to enhanced infection by protists. A total of 210 TUGs contain one or more microsatellites as potential candidates for gene-linked genetic markers. Data are publicly available in a user-friendly database at http://www.uni-muenster.de/Evolution/ebb/Services/zostera

Peptidyl-prolyl cis-trans isomerase from Bacillus subtilis A prokaryotic enzyme that is highly sensitive to cyclosporin A

AbstractCyclophylins are members of a class of proteins with peptidyl-protyl cis-trans isomerase activity. These enzymes bind the immunosuppressive agent, cyclosporin A (CsA), which acts as a competitive inhibitor. The peptidyl-prolyl cis-trans isomerase from Bacillus subtilis (PPlase) was purified to homogeneity in a 4-step purification procedure, which resulted in a 100-fold protein purification with a yield of 5%, Coomassie blue-stained SDS-PAGE revealed a single band of about 18 kDa. PPlase activity was determined using synthetic peptides as substrates in a 2-step reaction coupled to chymotrypsin. Treatment of Bacillus subtilis PPlase by CsA revealed an inhibition constant of K1=175 nM, which differs from cyclophilin of enterobacteria such as E. coli or Salmonella typhimurium and is in the range of human enzymes