Abstract
Zygospores of green alga such as Chlamydomonas reinhardtii, Volvox carteri or Dunaliella salina display a bright orange color indicative of carotenoids, yet there have been no reports on their pigment composition. The genomes of these algae contain genes for homologs of the β-carotene ketolase (BKT) from the well-known astaxanthin producer Haematococcus pluvialis, that were assumed to be pseudogenes, because none of these species has been reported to accumulate astaxanthin or other ketocarotenoids. Here, we show that C. reinhardtii and V. carteri synthesize ketocarotenoids specifically in zygospores. Contrary to the vegetative aplanospores of H. pluvialis, the major ketocarotenoid in zygospores of C. reinhardtii is not astaxanthin but 4-ketolutein. Moreover, the ketocarotenoids in maturing zygospores are not synthesized de novo but from carotenoids of the photosynthetic apparatus liberated by a massive breakdown of thylakoid membranes. In line with this conclusion, incubation of zygospores at 9°C instead of 22°C resulted in a reduced thylakoid breakdown and only low amounts of ketocarotenoids, while the accumulation of storage lipids was less affected. Furthermore, we show the full-length BKT from C. reinhardtii to catalyze the ketolation of both α-carotene and lutein in carotenogenic bacteria. We also detected putative BKT genes in the genomes of various other green algae not yet known to synthesize ketocarotenoids, suggesting a zygospore-specific accumulation of ketocarotenoids to be common among Chlamydomonadales. Our observation that zygospores of C. reinhardtii accumulate ketocarotenoids together with storage lipids sheds light on the physiology of a largely unexplored algal life stage crucial for survival and propagation.