More recently, C. vulgaris NJ-7, a strain isolated from the Antarctic, was used to investigate the adaptation of eukaryotic microbes to permanently cold environments (Hu et al., 2008; Li et al., 2009). The strain NJ-7 possesses the same 18S rRNA gene sequence as that of UTEX259, a strain isolated

from the temperate region, but shows a significantly intensified freezing tolerance (5- to  1000-fold higher viability) than the temperate strain. Comparative studies of the two C. vulgaris strains provide opportunities to understand how intra-species learn more evolution is undertaken in eukaryotic microbes to adapt to the Antarctic or other extreme environments. HIC6 is a group-3 late embryogenesis abundant (LEA) protein found in C. vulgaris. Together with HIC12, it was first identified by 2D-HPLC and SDS-PAGE to be hardening (cold treatment)-induced in the strain C-27 (Honjoh et al., 1995). Its cDNA was also identified by differential screening of a cDNA library (Joh et al., 1995) or suppression subtractive hybridization (Machida et al., 2008). LEA Rapamycin mouse proteins were initially found at the late stage of embryogenesis in cotton (Galau et al., 1986) and were subsequently found in algae (such as C. vulgaris), cyanobacteria (Close & Lammers, 1993), nematodes (Browne

et al., 2002) and fungi (Abba’ et al., 2006). The proteins can be divided into different groups on the basis of similarities in amino acid sequences (Colmenero-Flores et al., 1997; Cuming, 2005; Battaglia et al., 2008). Like many other LEA proteins, HIC6 remained soluble under boiling conditions and showed in vitro cryoprotective activities on lactate dehydrogenase (LDH) (Honjoh et al., 2000). Overexpression of HIC6 in plant or yeast could enhance their freezing tolerance (Honjoh et al., 1999, 2001), and

in the transgenic plant, HIC6 was localized to mitochondria (Honjoh et al., 2001). In strains NJ-7 and UTEX259, the encoding gene hiC6 was also induced upon exposure to cold, and the expression was intensified in strain NJ-7 in comparison with UTEX259 (Li et al., 2009). These results suggest that the enhanced expression of hiC6 is probably involved in the development of freezing tolerance in C. vulgaris. The intensified expression of hiC6 in NJ-7 could be due to gene duplication, increased transcription or post-transcriptional regulation. Obatoclax Mesylate (GX15-070) In our previous study, only one hiC6 gene was identified in each of the two Chlorella strains, NJ-7 and UTEX259 (Li et al., 2009). In the present study, however, sequencing of that chromosomal region revealed that multiple hiC6 genes are organized in tandem in both strains. The tandem-arrayed genes encode different HIC6 isoforms and are differentially expressed. Chlorella vulgaris strains were grown in BG11 (Stanier et al., 1971) in the light of 50 μE m−2 s−1 at 20 °C with aeration. Cells grown at 20 °C were cooled to 4 °C in a water bath and transferred to a 4 °C refrigerator with aeration and illumination (50 μE m−2 s−1) for different periods of time.