Abstract
In Arabidopsis (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 is this is a major determinant of the low-temperature tolerance capacity of individual plant species? Here we have begun to address these questions through comparative analyses of Medicago truncatula and Medicago sativa subsp. falcata. M. truncatula survived to -4 degrees C but did not cold acclimate, whereas Medicago falcata cold acclimated and survived -14 degrees C. 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 C-REPEAT/DEHYDRATION RESPONSIVE ELEMENT (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 comprising 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, their proximity to one another is likely key to regulatory output in Medicago, and 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.
Highlights
In Arabidopsis (Arabidopsis thaliana) the low-temperature induction of genes encoding the C-REPEAT BINDING FACTOR (CBF) transcriptional activators is a key step in cold acclimation
As the CBF transcriptional activators play a central role in the cold-acclimation response of Arabidopsis, we considered that M. truncatula and M. falcata might differ in one or more components of the CBF response pathway
The 5-bp C-REPEAT/DEHYDRATION RESPONSIVE ELEMENT (CRT/DRE) motif forms the conserved core of a cis-acting regulatory element to which the CBF transcription factors bind (Baker et al, 1994; YamaguchiShinozaki and Shinozaki, 1994; Stockinger et al, 1997; Liu et al, 1998)
Summary
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China. The presence of the clustered CRT/DRE and ABRE motifs in the distal upstream conserved block of M. truncatula and M. falcata CAS15 suggested that this region might function as a regulatory island critical for the low-temperature induction of CAS15. To explore this idea we generated MtCAS15 promoter constructs that were either inclusive of the CRT/DRE and ABRE motifs (pMC7), or exclusive of these motifs (pMC1), fused the promoter regions to the uidA (GUS) reporter gene, and transformed them into Arabidopsis (Fig. 5; Supplemental Fig. S1). A single M. falcata accession harbors two allelic forms of these genes
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