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Published on January 24, 2008; 10.1104/pp.107.108779

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Received September 8, 2007
Accepted January 22, 2008

Comparative genomic sequence and expression analyses of Medicago truncatula and M. sativa ssp. falcata cold-acclimation specific (CAS) genes

Joyce C. Pennycooke , Hongmei Cheng , and Eric J. Stockinger *

Department of Horticulture and Crop Science, 1680 Madison Avenue, The Ohio State University/OARDC, Wooster OH 44691; (Present address) Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China; Department of Horticulture and Crop Science, 1680 Madison Avenue, The Ohio State University/OARDC, Wooster OH 44691

* Corresponding author; email: stockinger.4{at}osu.edu.

In Arabidopsis thaliana the low-temperature induction of genes encoding the C-Repeat Binding Factor (CBF) transcriptional activators is a key step in cold acclimation. CBFs in turn activate a battery of downstream genes known as the CBF regulon, which collectively act to increase tolerance to low temperatures. Fundamental questions are what determines the size and scope of the CBF regulon, and whether this is a major determinant of the low temperature tolerance capacity of individual plant species. Here we've begun to address this question through comparative analyses of Medicago truncatula and M. sativa ssp. falcata. M. truncatula survived to -4oC but did not cold acclimate, whereas M. falcata cold acclimated and survived -14oC. Both species possessed low-temperature induced CBFs but differed in the expression of the cold acclimation specific, CAS genes which are candidate CBF targets. M. falcata CAS30 was robustly cold-responsive whereas the MtCAS31 homolog was not. M. falcata also possessed additional CAS30 homologs in comparison to the single CAS31 gene in M. truncatula. MfCAS30 possessed multiple pairs of closely-spaced CRT/DRE motifs, the cognate CBF binding site in its upstream region whereas MtCAS31 lacked one CRT/DRE partner of the two proximal partner-pairs. CAS genes also shared a promoter structure comprised of modules proximal and distal to the coding sequence. CAS15, highly cold responsive in both species, harbored numerous CRT/DRE motifs, but only in the distal module. However fusion of the MtCAS15 promoter, including the distal module, to a reporter gene did not result in low temperature responsiveness in stably transformed Arabidopsis. In contrast, both MtCAS31 and MfCAS30 promoter fusions were low temperature responsive, although the MfCAS31 fusion was less robust than the MfCAS30 fusion. From these studies we conclude that CAS genes harbor CRT/DRE motifs, that their proximity to one another is likely key to regulatory output in Medicago, and that they may be located kilobases distal to the transcriptional start site. We hypothesize that these differences in CRT/DRE copy numbers in CAS30/CAS31 upstream regions combined with differences in gene copy numbers may be a factor in determining differences in low temperature tolerance between M. truncatula and M. falcata.






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