Hardening-induced Chlorella Genes and Proteins

Seventeen cDNA clones corresponding to hardening-induced Chlorella (hiC) genes were isolated by differential screening of a cDNA library from 6 h-hardened cells of Chlorella vulgaris C-27, a frost-hardy strain. Northern analysis showed that all hiC transcripts are specific to low temperature and their induction patterns are diverse. Genomic Southern and dot analyses of 2 strains of Chlorella (C. vulgaris C-27, frost-hardy; C. ellipsoidea C-102, chilling-sensitive) revealed that 10 out of 17 hiC genes are not detected in C-102 and the other 7 hiC genes were not expressed in C-102 except only one gene. The expression of these 16 genes is probably necessary for the acquisition of freezing tolerance. Homology searches of the sequences of the hiC clones resulted in the following data: hid, chaperonin-60β polypeptide; hiC2, desiccation-related protein; hiC3, thioredoxin peroxidase; hiC4, ribosomal protein L 2; hiC6, group III LEA (Late Embryogenesis Abundant) protein; hiC7, peptidyl-prolyl cis-trans isomerase; hiC8, ribosomal protein S-27; hiCIO, zeste protein; hiC12, group III LEA protein (different from hiC6). On the other hand, 31 soluble proteins, which were induced or increased during hardening, were isolated by 2D-HPLC and SDS-PAGE. The N-terminal sequences of 7 out of 31 proteins were determined. The results of homology search were as follows : 14-kDa and 10-kDa proteins (identical with HIC6 and HIC12 proteins, respectively), group III LEA proteins; 22-kDa protein, thioredoxin peroxidase ; 18-kDa and 16.5-kDa proteins, photosystem II oxygen-evolving enhancer protein 2 ; the two other, no homology. The results obtained showed that the LEA protein (HIC 6) is highly involved in the development of frost hardiness. Introduction of the lea gene enhanced freezing tolerance in yeast.