Dissecting the Heat Stress Response in Chlamydomonas by Pharmaceutical and RNAi Approaches Reveals Conserved and Novel Aspects

To study how conserved fundamental concepts of the heat stress response （HSR） are in photosynthetic eukaryotes, we applied pharmaceutical and antisense/amiRNA approaches to the unicellular green alga Chlamydomonas reinhardtii. The Chlamydomonas HSR appears to be triggered by the accumulation of unfolded proteins, as it was induced at ambient temperatures by feeding cells with the arginine analog canavanine. The protein kinase inhibitor staurosporine strongly retarded the HSR, demonstrating the importance of phosphorylation during activation of the HSR also in Chlamydomonas. While the removal of extracellular calcium by the application of EGTA and BAPTA inhibited the HSR in moss and higher plants, only the addition of BAPTA, but not of EGTA, retarded the HSR and impaired thermotoler- ance in Chlamydomonas. The addition of cycloheximide, an inhibitor of cytosolic protein synthesis, abolished the attenu- ation of the HSR, indicating that protein synthesis is necessary to restore proteostasis. HSP90 inhibitors induced a stress response when added at ambient conditions and retarded attenuation of the HSR at elevated temperatures. In addition, we detected a direct physical interaction between cytosolic HSP90A/HSP70A and heat shock factor 1, but surprisingly this interaction persisted after the onset of stress. Finally, the expression of antisense constructs targeting chloroplast HSP70B resulted in a delay of the cell＇s entire HSR, thus suggesting the existence of a retrograde stress signaling cascade that is desensitized in HSP7OB-antisense strains.

PETO Interacts with Other Effectors of Cyclic Electron Flow in Chlamydomonas

While photosynthetic linear electron flow produces both ATP and NADPH, cyclic electron flow （CEF） around photosystem I （PSI） and cytochrome bef generates only ATP. CEF is thus essential to balance the supply of ATP and NADPH for carbon fixation; however, it remains unclear how the system tunes the relative levels of linear and cyclic flow. Here, we show that PETO, a transmembrane thylakoid phosphoprotein specific of green algae, contributes to the stimulation of CEF when cells are placed in anoxia. In oxic conditions, PETO co-fractionates with other thylakoid proteins involved in CEF （ANR1, PGRL1, FNR）. In PETO-knock- down strains, interactions between these CEF proteins are affected. Anoxia triggers a reorganization of the membrane, so that a subpopulation of PSi and cytochrome bsf now co-fractionates with the CEF effectors in sucrose gradients. The absence of PETO impairs this reorganization. Affinity purification identifies ANR1 as a major interactant of PETO. ANR1 contains two ANR domains, which are also found in the N-terminal region of NdhS, the ferredoxin-binding subunit of the plant ferredoxin-plastoquinone oxidoreductase （NDH）. We propose that the ANR domain was co-opted by two unrelated CEF systems （PGR and NDH）, possibly as a sensor of the redox state of the membrane.