Cold-Induced Changes in Freezing Tolerance, Protein Phosphorylation, and Gene Expression (Evidence for a Role of Calcium)

Abstract

l h e role of Ca'+ in cold-induced changes i n protein phosphorylation, gene expression, and development of freezing tolerance has been studied in cell-suspension cultures of a freezing-tolerant cultivar of alfalfa (Medicago safiva spp. falcafa cv Anik). Chemical treatments to block Caz+ channels, antagonize calmodulin action, or inhibit protein kinases markedly inhibited the cellular capacity to develop cold-induced freezing tolerance but had little effect on cell viability. An analysis of phosphoprotein profile by two-dimensional polyacrylamide gel electrophoresis revealed that at low temperature the relative level of phosphorylation of several proteins increased, whereas that of several others decreased. When cold acclimation was carried out in the presence of M(6-aminohexyl)-5-chloro-l-naphthalene-sulfonamide hydrochloride, an antagonist of calmodulin and ca'+-dependent protein kinases, or the CaZ+ channel blocker La3+, the cold-induced changes i n protein phosphorylation were strongly inhibited, cells lost their capacity to develop freezing tolerance, and accumulation of transcripts of cold acclimation-specific genes was substantially reduced. An inhibitor of protein kinases, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, had less pronounced effects on the cold-induced protein phosphorylation and caused only a partia1 inhibition of the cold-induced development of freezing tolerance and accumulation of the transcripts. l h e level of phosphorylation of one protein, of about 15 kD, increased more than 10-fold at low temperature and showed a strong positive correlation with cold-induced freezing tolerance and gene expression even when the latter were altered with various chemical treatments. lhese results suggest that CaZ+ and protein phosphorylation, or perhaps a coupling of the two, play an important role during the acquisition of freezing tolerance during cold acclimation.

A Calcium/Calmodulin-regulated Member of the Receptor-like Kinase Family Confers Cold Tolerance in Plants

“…Isolation of CRLK1-interacting proteins/substrates will help in further dissecting this signal pathway. Accumulating evidence indicates that protein phosphorylation is involved in the pathway connecting the cold-triggered calcium changes and cold acclimation (22,39). However, the protein kinase(s) responsible for inducing cold-regulated genes and activating freezing tolerance is elusive.…”

Roles forArabidopsisCAMTA Transcription Factors in Cold-Regulated Gene Expression and Freezing Tolerance

“…In Arabidopsis, the rapid influx of calcium into the cytosol is required for normal cold induction of the CBF target genes KIN1 and KIN2 (Knight et al, 1996;Tahtiharju et al, 1997). Similarly, in alfalfa, cold-induced accumulation of transcripts for two coldresponsive genes, cas15 and cas18, is dependent upon calcium (Monroy et al, 1993;Monroy and Dhindsa, 1995).…”

Molecular Basis of Plant Cold Acclimation: Insights Gained from Studying the CBF Cold Response Pathway

“…Nearly 20 years ago, Knight et al (1991) established that exposure of plants to low temperature results in a rapid transient spike in cytoplasmic calcium levels. Subsequent studies linked the cold-induced calcium spikes to cold-regulated gene expression in Arabidopsis (Knight et al, 1996;Tahtiharju et al, 1997;Henriksson and Trewavas, 2003) and alfalfa (Medicago sativa; Monroy et al, 1993;Monroy and Dhindsa, 1995). For instance, the studies with Arabidopsis demonstrated that chemical agents that reduce calcium influx into the cytoplasm impaired cold induction of certain CBF regulon genes.…”