Modulation of TOR kinase activity inChlamydomonas reinhardtii: Effect of N-starvation and changing carbon pool

Abstract

Target Of Rapamycin (TOR) kinase is a sensor as well as a central integrator of internal and external metabolic cues. The upstream and downstream signals of this kinase are very well characterized in animals. However, in algae, higher plants and other photosynthetic organisms, the components of the TOR kinasesignaling are yet to be characterized. Here, we establish an assay system to study TOR kinase activity in C.reinhardtii using the phosphorylation status of its downstream target, CrS6K. We further use this assay to monitor TOR kinase activity under various physiological states such as photoautotrophy, heterotrophy, mixotrophy and nitrogen starvation. We observe that autotrophy in light (and not in dark) leads to TOR kinase attenuation during N starvation while the same is not observed in mixotrophy. Importantly, we show that the external carbon source glucose is sensed and uptaken by C.reinhardtii cells only in the presence of light and not in the dark. And such exogenously added glucose, as the photoassimilate carbon mimic, results in enhanced production of ROS, induction of autophagy and concomitant drop in TOR kinase activity, creating Nstarvation-like cellular state even in N+ conditions. Interestingly, dose dependent addition of glucose revealed TOR kinase activation in low glucose regime (ROS independent) followed by attenuation of TOR kinase (ROS dependent) at high glucose levels.

Autophagy induced accumulation of lipids inpgrl1andpgr5ofChlamydomonas reinhardtiiunder high light

“…The total ROS was detected with 2,7dichlorodihydrofluorescein diacetate (H2DCFDA), a fluorescence dye to detect the ROS in live Chlamydomonas cells. Cells at a 3 million density were collected from all the conditions, and H2DCFDA staining was performed at 10 µM concentration (Upadhyaya et al, 2018). Further, cells were incubated with dye for 1h at RT in a continuously rotating shaker under dark.…”

Metabolic responses to nitrogen deficiency and rapamycin treatment in freshwater microalgae strains

“…Recently, it was identified a single copy of CrS6K in C. reinhardtii and confirmed the presence of the well conserved TOR phosphorylation motif FLGFTYVAP at the C-terminal of the protein (Upadhyaya et al, 2018). It still has not been clear whether this protein is a canonical S6K, although the C-terminus of the protein contains the TOR phosphorylation motif.…”

“…The sensitivity of Chlamydomonas to rapamycin has been considered an experimental advantage to explore the function of TOR in a photosynthetic organism (Dobrenel et al, 2016a;Upadhyaya et al, 2018;Mubeen et al, 2019;Prioretti et al, 2019). The mechanism by which TOR regulates transcription is still poorly understood not only in microalgae but also in land plants and is a topic to be addressed in future research.…”

“…It still has not been clear whether this protein is a canonical S6K, although the C-terminus of the protein contains the TOR phosphorylation motif. Still according to Upadhyaya et al, (2018), CrS6K is involved in protein synthesis under light conditions. Therefore, it has been reported no direct evidence relating to the involvement of CrS6K in protein synthesis.…”

“…Chloroplasts are one of the most important cellular compartments of plant and algae cells, as they are responsible for the essential processes of photosynthesis (Couso et al, 2021). The availability of nutrients and other environmental conditions determine the size, number and activity of chloroplasts in plant cells (Li et al, 2013;Upadhyaya et al, 2018;Ingargiola et al, 2020). How the regulation of chloroplast processes occurs via TOR remains largely unknown.…”