Cloning, Characterization, and Expression of a cDNA Encoding a 50-Kilodalton Protein Specifically Induced by Cold Acclimation in Wheat

Abstract

We have isolated, sequenced, and expressed a cold-specific cDNA clone, Wcs120, that specifically hybridizes to a major mRNA species of approximately 1650 nucleotides from cold-acclimated wheat (Triticum aestivum L.). The accumulation of this mRNA was induced in less than 24 hours of cold treatment, and remained at a high steady-state level during the entire period of cold acclimation in the two freezing-tolerant genotypes of wheat tested. The expression of Wcs120 was transient in a less-tolerant genotype even though the genomic organization of the Wcs120 and the relative copy number were the same in the three genotypes. The mRNA level decreased rapidly during deacclimation and was not induced by heat shock, drought, or abscisic acid. The Wcs120 cDNA contains a long open reading frame encoding a protein of 390 amino acids. The encoded protein is boiling stable, highly hydrophilic, and has a compositional bias for glycine (26.7%), threonine (16.7%), and histidine (10.8%), although cysteine, phenylalanine, and tryptophan were absent. The WCS120 protein contains two repeated domains. Domain A has the consensus amino acid sequence GEKKGVMENIKEKLPGGHGDHQQ, which is repeated 6 times, whereas domain B has the sequence TGGTYGQQGHTGTT, which is repeated 11 times. The two domains were also found in barley dehydrins and rice abscisic acidinduced protein families. The expression of this cDNA in Escherichia coli, using the T7 RNA polymerase promoter, produced a protein of 50 kilodaltons with an isoelectric point of 7.3, and this product comigrated with a major protein synthesized in vivo and in vitro during cold acclimation.

Identification and characterization of a low temperature regulated gene encoding an actin‐binding protein from wheat

“…Analysis and sequence comparisons were carried out with the Genetic Computer Group's sequence analysis software package, version 6.0. RNA and DNA gel blot analysis were performed as described previously [8].…”

Differential expression of a gene encoding an acidic dehydrin in chilling sensitive and freezing tolerant gramineae species

“…The pWcor410 clone was isolated from a Lambda Zap II library constructed from poly(A)' RNA isolated from cold-acclimated winter wheat (1: aestivum L. cv Norstar) [9]. Differential screening of this library was done with cDNA probes synthesized from poly(A)' RNA isolated from control and cold-acclimated winter wheat.…”

“…DNA sequence was determined on both strands. All other molecular biology techniques were performed using standard procedures [lo] and modifications already published [9].…”

A cold-responsive wheat (Triticum aestivum L.) gene wcor14 identified in a winter-hardy cultivar 'Mironovska 808'.

“…A gene wcs120 and its related, perhaps paralogous genes including Wcor39, Wcs66, Wcor80, Wcor200 and Wcor726 are expressed in response to low temperature (Houde et al, 1992;Guo et al, 1992;Quellet et al, 1993;Chauvin et al, 1994;Danyluk et al, 1996;Hughes and Dunn, 1996). Among them, wcs120 and wcor200 are the most well characterized members respectively encoding glycine-rich neutral proteins of 50 kDa (pI = 7.3) and 200 kDa (pI = 6.5) with characteristics of the group 2 LEA (or D-11) family, although they do not have a conserved polyserine sequence (Houde et al, 1992;Quellet et al, 1993). wcor410 encodes an acidic (pI = 5.1) DHNlike group 2 LEA protein of 28 kDa with 15-mer lysinerich motifs and a polyserine sequence (Danyluk et al, 1994).…”