PHOTOCHEMICAL CHARACTERIZATION OF CHLAMYDOMONAS MUTANTS FOR HERBICIDE PERCEPTION

Much research efforts are focused on implementation of sensor applications exploiting photosynthetic bio-recognition elements mostly due to the high susceptibility of the primary photosynthetic reactions towards variety of chemical species [1]. Photosynthetic microalgae are often among the preferred sensing elements in biosensor devices for large-scale and in field monitoring of different classes of pesticides or heavy metals. The current challenges in using photosynthetic bio-recognition element are mainly related to improving the stability and sensitivity of the biological component. The ultimate goal of this research is the realization of robust, sensitive, and reliable photosynthetic sensing elements by altering the aminoacid composition of the D1 protein of photosystem II (PSII) in the model algae Chlamydomonas reinhardtii. Taking advantage of protein engineering techniques and computational analyses it was possible to identify a set of single point mutations in D1 conferring improved tolerance to free-radicals-associated stress and/or competence for herbicide perception [2, 3]. Particularly, this study is focused on photochemical characterisation of Chlamydomonas strains hosting a substitution of D1-Ile163 with Asn (I163N) and D1-Phe265 with Thr (F256T) or Ser (F265S) by registering thermoluminescence (TL) glow curves and kinetics of oxygen-evolving reactions. Both methods provide comprehensive information about the efficiency of the PSII photochemistry and offer useful criteria for the functional characterisation step of the bio-recognition element. The results indicated that the mutation introduced near to the PSII redox-active Tyr161 (I163N) may stabilize PSII charge separation, while the alteration in the composition of the QB binding pocket (F256T and F265S) lead to noticeable impairment of the QA to QB electron transfer. Peculiarities of the PSII performance in selected strains and their suitability as bio-recognition elements will be presented.