Functional in vivo Studies of Algal Sensory Photoreceptors

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The interplay of the Chlamydomonas Cryptochromes aCry and pCry for time setting of the endogenous clock, surface attachment and gliding, and high energy quenching is still an open question. We will focus on the inactivation of aCRY, pCRY and a hypothetical red-light photoreceptor in different strains and study the influence of these receptors on several physiological responses. We will grow cells under different oxygen, light, and nutrient conditions, apply different illumination programs, and monitor physiological responses as well as translation and protein abundance in wt and deletion strains and study the influence on photosynthesis and high energy quenching. We will focus on the abundance of clock proteins, accumulation of the light harvesting protein LHCSR3 and of other photoreceptor proteins as channelrhodopsins, histidine-kinase-rhodopsins (HKRs), and phototropin (Phot). We will study internal Ca2+-signaling including Ca2+ release from Chloroplast and ER. In a second approach we will study the in vivo function of Bestrhodopsins, which are novel hybrids of rhodopsins with bestrophin domains forming large complex light-sensitive ion channels comprising 5 channel subunits and 5 or 10 rhodopsins. We will characterize the bestrhodopsin in vivo function by replacing in Chlamydomonas the three light-insensitive bestrophins with the light modulated bestrhodopsins from Cymbomonas analyze the function within the cellular and organismal context. We consider this as an ideal and complementary approach to better understand the in vivo function of both bestrophin and bestrhodopsin at the same time.