Pennycooke Joyce C, Cheng Hongmei, Stockinger Eric J
Department of Horticulture and Crop Science, The Ohio State University/Ohio Agricultural Research and Development Center, Wooster, OH 44691, USA.
Plant Physiol. 2008 Mar;146(3):1242-54. doi: 10.1104/pp.107.108779. Epub 2008 Jan 24.
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.
在拟南芥中，编码C-重复序列结合因子（CBF）转录激活因子的基因的低温诱导是冷驯化的关键步骤。CBF反过来激活一系列被称为CBF调控子的下游基因，这些基因共同作用以提高对低温的耐受性。基本问题是：什么决定了CBF调控子的大小和范围，以及这是否是单个植物物种低温耐受能力的主要决定因素？在这里，我们开始通过对蒺藜苜蓿和黄花苜蓿亚种进行比较分析来解决这些问题。蒺藜苜蓿能在-4℃存活但不能进行冷驯化，而黄花苜蓿能进行冷驯化并能在-14℃存活。两个物种都具有低温诱导的CBF，但在冷驯化特异性（CAS）基因的表达上存在差异，这些基因是CBF的候选靶标。黄花苜蓿的CAS30对低温有强烈响应，而蒺藜苜蓿的MtCAS31同源物则没有。与蒺藜苜蓿中的单个CAS31基因相比，黄花苜蓿还拥有额外的CAS30同源物。MfCAS30在其上游区域拥有多对紧密间隔的C-重复序列/脱水响应元件（CRT/DRE）基序，即同源CBF结合位点，而MtCAS31缺少两个近端伴侣对中的一个CRT/DRE伴侣。CAS基因还共享一个启动子结构，该结构由编码序列近端和远端的模块组成。在两个物种中对低温都有高度响应的CAS15含有大量CRT/DRE基序，但仅存在于远端模块中。然而，将包括远端模块的MtCAS15启动子与一个报告基因融合，在稳定转化的拟南芥中并未导致低温响应。相比之下，MtCAS31和MfCAS30启动子融合都具有低温响应性，尽管MfCAS31融合的响应性不如MfCAS30融合。从这些研究中我们得出结论，CAS基因含有CRT/DRE基序，它们彼此的接近程度可能是蒺藜苜蓿中调控输出的关键，并且它们可能位于转录起始位点数千碱基的远端。我们假设，CAS30/CAS31上游区域中CRT/DRE拷贝数的这些差异与基因拷贝数的差异可能是决定蒺藜苜蓿和黄花苜蓿低温耐受性差异的一个因素。
