Abstract
A cold-responsive chitinase gene, BiCHT1, was isolated from bromegrass (Bromus inermis) 'Manchar' suspension cells. BiCHT1 messenger RNA was detected at low levels in nonstressed bromegrass cells, whereas its accumulation was induced by incubation at 10 degrees C and 4 degrees C as detected by northern- and western-blot analyses. BiCHT1 was highly homologous to rye CHT9, known to encode an antifreeze protein. BiCHT1 was overexpressed in Escherichia coli and bromegrass cells using genetic transformation procedures. BiCHT1 products expressed in both systems had chitinase activity, but the expressed proteins did not affect the growth of ice crystals in any conditions tested. Besides cold stress, the expression of the BiCHT1 gene was up-regulated by exposure to 35 degrees C, but not by salt or osmotic stress, abscisic acid, or ethephon. BiCHT1 messenger RNA did not accumulate in response to methyl jasmonate and salicylic acid, but was slightly increased by prolonged culture at 25 degrees C and only transiently by chitin. Antifreeze activity detected in the culture medium was induced at 4 degrees C but only slightly at 10 degrees C. It was also induced by ethephon treatment, but not by abscisic acid, chitin, or prolonged incubation at 25 degrees C. The results of transgenics and expression analyses suggest that the BiCHT1 product is a major protein with chitinase activity secreted in the medium of cold-treated cells and is unlikely to be responsible for the antifreeze activity detected in the culture medium.
Highlights
A cold-responsive chitinase gene, BiCHT1, was isolated from bromegrass (Bromus inermis) ‘Manchar’ suspension cells
BiCHT1 was highly homologous to winter rye CHT9, known to encode an antifreeze proteins (AFPs) (Yeh et al, 2000)
BiCHT1 cDNAwas cloned from mRNA isolated from cold-treated bromegrass cells as previously described (Nakamura et al, unpublished data)
Summary
A cold-responsive chitinase gene, BiCHT1, was isolated from bromegrass (Bromus inermis) ‘Manchar’ suspension cells. The results of transgenics and expression analyses suggest that the BiCHT1 product is a major protein with chitinase activity secreted in the medium of cold-treated cells and is unlikely to be responsible for the antifreeze activity detected in the culture medium. AFPs inhibit ice recrystallization (Griffith and Yaish, 2004), which reduces ice crystal sublimation and maintains small ice crystals in frozen tissues This prevents ice crystals from growing into tissue-damaging sizes during prolonged exposure to subzero temperatures and is thought to improve winter survival in the field (Chun et al, 1998). Based on these properties, AFPs are thought to play important roles in freezing tolerance and overwintering mechanisms.
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