Comparative Genomic Sequence and Expression Analyses ofMedicago truncatulaand Alfalfa Subspeciesfalcata COLD-ACCLIMATION-SPECIFICGenes    

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°C but did not cold acclimate, whereas Medicago falcata cold acclimated and survived −14°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.

Opposing control by transcription factors MYB61 and MYB3 Increases Freezing Tolerance by relieving C-repeat Binding Factor suppression

“…MtCAS15 (Medtr7g086340) is induced by low temperature in Medicago spp., and a positive correlation between freezing tolerance and its expression level was identified in Medicago spp. (Monroy et al, 1993a;Pennycooke et al, 2008;Zhang et al, 2011). In addition, MtLTI65 (AC235674_16) and MtY3G1 (Medtr2g014050; a late embryogenesis abundant protein) also were reported to participate in cold stress in previous studies (Nordin et al, 1993;Hincha and Thalhammer, 2012).…”

“…The freezing tolerance assay in M. truncatula was executed as described previously (Pennycooke et al, 2008) with several modifications. Briefly, 5-week-old whole plants grown in a soil:vermiculite (1:2, v/v) mixture with or without cold acclimation were subsequently placed in a freezing chamber (RuMED4001; Shi et al, 2012) that was set to maintain a temperature of 0°C for 1 h. The temperature was then decreased at a rate of 1°C h 21 in the dark.…”

“…However, Zhang et al (2011) demonstrated that M. truncatula is able to increase its freezing tolerance after being subjected to cold-acclimating conditions. The different experimental conditions and methods used in these two studies may partly explain the different results (Pennycooke et al, 2008;Zhang et al, 2011). By examining physiological markers of cold acclimation in M. truncatula, Thapa (2008) also independently demonstrated that this species undergoes cold acclimation.…”

“…In alfalfa (Medicago sativa), cold induces a calcium influx, which affects protein phosphorylation and the expression levels of cold acclimation-specific genes such as CAS15 and CAS18 (Mohapatra et al, 1989;Monroy et al, 1993aMonroy et al, , 1993bWolfraim et al, 1993;Monroy and Dhindsa, 1995). In M. truncatula and Medicago falcata, cold induces the expression of CBF1 to CBF3 and CAS genes (Pennycooke et al, 2008;Zhang et al, 2011). The expression level of MtCBF4 is induced by drought, salinity, and cold stress, and ectopic MtCBF4 expression in Arabidopsis enhances drought and salt tolerance, as well as salt tolerance when transiently expressed in M. truncatula roots (Li et al, 2011).…”

“…Many plants increase their freezing tolerance via a process known as cold acclimation, which involves undergoing a period of low but nonfreezing temperatures (Thomashow, 1999). Pennycooke et al (2008) reported that Medicago truncatula does not cold acclimate. However, Zhang et al (2011) demonstrated that M. truncatula is able to increase its freezing tolerance after being subjected to cold-acclimating conditions.…”