Overexpression of Orange (OR) and OR mutant protein in Chlamydomonas reinhardtii enhances carotenoid and ABA accumulation and increases resistance to abiotic stress

The carotenoid content of plants can be increased by overexpression of the regulatory protein ORANGE (OR) or a mutant variant known as the golden SNP. In the present study, transgenic lines of the microalgae Chlamydomonas reinhardtii were generated to overexpress either wild type CrOR (CrORWT) or a mutated CrOR (CrORHis) containing a single histidine substitution for a conserved arginine. Overexpression of both CrORWT and CrORHis dramatically enhanced the accumulation of several different carotenoids, including {beta}-cartotene, -carotene, lutein and violaxanthin, in C. reinhardtii and, in contrast to higher plants, upregulated the transcript abundance of several relevant carotenoid biosynthetic genes. In addition, microscopic analysis revealed that the OR transgenic cells were larger than control cells and exhibited larger chloroplasts with a disrupted morphology. Moreover, both CrORWT and CrORHis cell lines showed increased tolerance to salt and paraquat stress. The levels of endogenous phytohormone abscisic acid (ABA) were also increased in CrORWT and CrORHis lines, not only in normal growth conditions but also in growth medium supplemented with paraquat. Together these results offer new insights regarding the role of the OR protein in regulating carotenoid biosynthesis and accumulation in microalgae, and establish a new functional role for OR to modulate oxidative stress tolerance mediated by ABA.