In notothenioid fish, gene duplications that enhance gene expression play an important role in adaptation to the Antarctic environments (Chen et al., 2008). As we found a slightly higher copy number of hiC6 in NJ-7 than UTEX259, it would be interesting to isolate more C. vulgaris

strains with a lower freezing tolerance and determine whether the copy number of hiC6 would decrease to one to about three accordingly. We also wondered whether all copies of hiC6 in the tandem array were identically expressed. Because hiC6 genes have almost identical mRNA sequences, their expression can barely be distinguished by Northern blot hybridization. We employed gene-specific primers to perform RT-PCR detections and, in addition, calculated the relative transcript abundance based on sequences of total hiC6 MK 2206 cDNAs. Our results showed that the tandem-arrayed genes were differentially expressed in both strains. In NJ-7, almost all hiC6 transcripts were expressed from NJ7hiC6-3, -4 and -5, whereas in UTEX259, hiC6 transcripts were essentially expressed from 259hiC6-1, -3 and -4. Therefore, the formation of the tandem array

of hiC6 does not appear to be a simple process of gene duplications but takes place in combination with gene expression divergence. In an Antarctic green alga species, the nitrate reductase showed a lower maximal temperature compared to that of a temperate Acalabrutinib species (di Rigano et al., 2006). This finding suggests that proteins can be evolved to promote the adaptation to Antarctic environments. We wondered whether amino acid substitutions within HIC6 can enhance the freezing tolerance of C. vulgaris. In vitro assays showed that different HIC6 isoforms

provided similar protection of LDH from inactivation by freeze and thaw. Therefore, compared to changes in gene expression level, accumulation of substitutions to enhance the cryoprotective activities of LEA proteins is probably a much slower process for adaptation to the Antarctic environments. In addition to HIC6 and HIC12, we have very recently identified two novel cold-inducible LEA proteins, Ccor1 and Ccor2, in NJ-7 (Liu et al., 2011). Probably, more LEA clonidine proteins remain to be identified. These proteins may exert cumulative effects on the freezing tolerance of Chlorella. Alternatively, they may be involved in protection of enzymes or membranes of different cellular structures and play independent roles in freezing tolerance. For example, HIC6 seemed to be localized to mitochondria in transgenic plants (Honjoh et al., 2001). Further analyses of these LEA proteins and their encoding genes should be very useful for an in-depth understanding of the development of freezing tolerance in the Antarctic Chlorella. This research was supported by the Key Projects KSCX2-YW-G-060 and KSCX2-SW-332 of Knowledge Innovation Program of Chinese Academy of Sciences. “
“A self-subunit swapping chaperone is crucial for cobalt incorporation into nitrile hydratase.