Influence of cold hardening on chlorophyll and carotenoid in Chlorella vulgaris

Chlorella vulgaris C-27 has developed freezing tolerance by hardening at 3°C for 24 hour. It was reported that many genes which encode proteins related stress response, storage, protein synthesis and metabolism including ζ -carotene desaturase increase at transcriptional levels. We focus on the change of photosynthesis pigments, and assessed the effects of the cold acclimation on chlorophyll and carotenoid in the hardened and unhardened cells of C. vulgaris C-27 and C. vulgaris C-102 (a chilling-sensitive strain). Cold hardening at 3°C for 24 hour caused a slight reduce of the chlorophyll content in C. vulgaris C-27, but did a significant reduction of that in C. vulgaris C-102. In Chlorella cells, six major carotenoids, i.e., β -carotene, α-carotene, lutein, zeaxanthin, violaxanthin and neoxanthin, were detected by an original method for carotenoid determination of C. vulgaris C-27 and C. vulgaris C-102. It was possible to separate lutein and zeaxanthin by HPLC using cholester column. The carotenoid composition was highly influenced by the cold hardening. It was elucidated that total carotenoid was little affected by hardening at 3°C, but zeaxanthin content increased with decrease of violaxanthin content. On the other hand, incubation at 25°C after freeze-thaw restored the altered levels of both pigments to pre-hardening levels. This result suggests that freezing tolerance of C. vulgaris C-27 induced during cold acclimation have a close involvement of change in xanthophyll cycle which plays a significant role to relieve oxidative stress at freezing and thawing.