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

Abstract

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, a strong light-inducible promoter was used to overexpress either wild type CrOR (CrORWT) or a mutated CrOR (CrORHis) containing a single histidine substitution for a conserved arginine in the microalgae Chlamydomonas reinhardtii. Overexpression of CrORWT and CrORHis roughly doubled and tripled, respectively, the accumulation of several different carotenoids, including β-carotene, α-carotene, lutein and violaxanthin in C. reinhardtii and upregulated the transcript abundance of nearly all 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 salt and paraquat. Together these results offer new insights regarding the role of the native OR protein in regulating carotenoid biosynthesis and the accumulation of several carotenoids in microalgae, and establish a new functional role for OR to modulate oxidative stress tolerance potentially mediated by ABA.